Polymerase-tethered nucleotides for nucleic acid synthesis

ABSTRACT

Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

CROSS-REFERENCING

This application is a continuation of Ser. No. 17/571,529; filed: Jan.9, 2022, which is a continuation of Ser. No. 16/230,438; filed: Dec. 21,2018, which is a continuation of PCT/US17/39120; filed: Jun. 23, 2017,which claims the benefit of Ser. No. 62/354,635, filed on Jun. 24, 2016,which application is incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberDE-AC02-05SC11231 awarded by the US Department of Energy. The governmenthas certain rights in the invention.

BACKGROUND

The great majority of Next Generation Sequencing (NGS) performed todayis based on “sequencing by synthesis” (SBS), in which the sequence of aprimed template molecule is determined by a signal resulting fromstepwise incorporation of complementary nucleotides by a polymerase(Goodwin et al., Nat Rev Genet. 2016 May 17; 17(6):333-51). Currently,the most popular method for SBS employs fluorescent “reversibleterminator” nucleotides (RTdNTPs)—nucleotides that are chemicallymodified to block elongation by a polymerase once they are incorporatedinto a primer. Post-incorporation, the free RTdNTPs are removed, and theidentity of the added base is determined by a fluorescent signal fromthe incorporated nucleotide. Next, the fluorescent reporter andterminating group are removed from the incorporated nucleotide,rendering the primer non-fluorescent and ready for subsequent extensionby a polymerase. By repeating this cycle of template-dependentextension, detection, and deprotection, the sequence of the templatemolecule is inferred from the sequence of fluorescence signals.

Contemporary DNA synthesis begins with chemical synthesis of tens tohundreds of 50-200 nt oligonucleotides (oligos) using thephosphoramidite method (Beaucage and Caruthers, Tetrahedron Letters22.20 (1981): 1859-1862). These oligos are assembled into kilobase-sizedproducts that are then isolated, sequence-verified, and amplified forsubsequent recombination into the full-length target sequence ifnecessary (Kosuri and Church, Nature methods 11.5 (2014): 499-507.).Despite decades of incremental improvement, each chemical step ofoligonucleotide synthesis results in 0.5-1.0% unreacted (orside-reacted) products, and these small losses compound exponentially todecimate the yield of the full-length oligo. Since many oligos areassembled into each kilobase-sized product, the presence of even a smallfraction of erroneous oligos in the assembly reaction will result inmost products containing at least one error. State of the art genesynthesis techniques apply various “error correction” strategies toenrich for error-free oligos or assembly products, but erroneous oligosare reported to be “the most crucial factor in DNA synthesis protocolstoday” (Czar et al., Trends in biotechnology 27.2 (2009): 63-72).Furthermore, many biologically relevant sequences, such as those withrepetitive or structure-forming regions and/or high or low G/C contentare difficult if not impossible to construct via assembly ofoligonucleotides, impeding their use in research and engineering. Thusfar, there is no practical method for de novo DNA synthesis using apolymerase to extend a nucleic acid in a cyclic manner analogous to SBS.

Arguably the key advances that lead to the NGS revolution came from thedevelopment of reversible terminator deoxynucleoside triphosphates(RTdNTPs) that can be incorporated into DNA by a polymerase andreversibly terminate further dNTP addition. Improved systems enablingsingle nucleotide extensions of a growing nucleic acid could benefit SBSand enable practical enzymatic de novo DNA synthesis.

SUMMARY

Provided herein, among other things, is a conjugate comprising apolymerase and a nucleoside triphosphate, where the polymerase and thenucleoside triphosphate are covalently linked via a linker thatcomprises a cleavable linkage. A set of such conjugates, where theconjugates correspond to G, A, T (or U) and C is also provided. Methodsfor synthesizing a nucleic acid of a defined sequence are also provided.The conjugates can also be used for sequencing applications.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1A. Scheme for two-step cyclic nucleic acid synthesis usingpolymerase-nucleotide conjugates. In the first step, a conjugateelongates a DNA molecule using its linked dNTP moiety; in the secondstep the linkage between the polymerase and the elongated DNA moleculeis cleaved, deprotecting the DNA molecule for subsequent elongation.

FIG. 1B. Scheme for two-step cyclic nucleic acid synthesis usingTdT-dNTP conjugates comprising a TdT molecule site-specifically labeledwith a dNTP via a cleavable linker.

FIG. 2 . Co-crystal structure of TdT (PDB ID: 4127) with anoligonucleotide and dNTP annotated to indicate the position of apossible linker attaching the dNTP to the polymerase.

FIGS. 3A-3E. Chemical detail of a scheme for tethering dUTP to TdT anduse of the conjugate to elongate a nucleic acid.

FIG. 3A. Starting materials to produce the thiol-reactivelinker-nucleotide OPSS-PEG4-amino-allyl-dUTP.

FIG. 3B. Structure of the thiol-reactive linker-nucleotideOPSS-PEG4-amino-allyl-dUTP.

FIG. 3C. Polymerase-nucleotide conjugate prepared by labeling TdT withOPSS-PEG4-amino-allyl-dUTP.

FIG. 3D. Elongation of a DNA molecule by the polymerase-nucleotideconjugate.

FIG. 3E. Cleavage of the linkage between the elongated DNA molecule andTdT.

FIGS. 4A-4E. Chemical detail of a scheme for tethering dCTP to TdT basedon a light cleavable linker and use of the conjugate to elongate anucleic acid.

FIG. 4A. Starting materials to produce the thiol-reactivelinker-nucleotide BP-23354-propargylamino-dCTP.

FIG. 4B. Structure of the thiol-reactive, light cleavablelinker-nucleotide BP-23354-propargylamino-dCTP.

FIG. 4C. Polymerase-nucleotide conjugate prepared by labeling TdT withBP-23354-propargylamino-dCTP.

FIG. 4D. Elongation of a DNA molecule by the polymerase-nucleotideconjugate.

FIG. 4E. Cleavage of the linkage between the elongated DNA molecule andTdT.

FIGS. 5A-5E. Scheme for DNA synthesis with real-time error correctionusing fluorescent polymerase-nucleotide conjugates.

FIG. 5A. A reaction chamber is loaded with a single molecule of theprimer.

FIG. 5B. The primer is elongated by a conjugate.

FIG. 5C. The elongation reaction is confirmed by detection of thereporter moiety of the conjugate. If the reporter is not detected, theelongation reaction is repeated.

FIG. 5D. The primer is deprotected by cleavage of the linker, releasingthe polymerase and reporter.

FIG. 5E. The deprotection reaction is confirmed by lack of detection ofthe reporter moiety. If the reporter is not detected, the deprotectionreaction is repeated.

FIG. 6 . Schematic drawing of an integrated microfluidic device for DNAsynthesis using fluorescent polymerase-nucleotide conjugates detectibleby TIRF microscopy.

FIGS. 7A-7E. Scheme for DNA sequencing using fluorescentpolymerase-nucleotide conjugates.

FIG. 7A. A reaction chamber is loaded with a primer-template duplex.

FIG. 7B. The primer-template duplex is exposed to a mixture ofconjugates of the four nucleotides labeled by distinct fluorophores andis elongated by a conjugate complementary to the first template base tobe sequenced.

FIG. 7C. The template base is identified by detection of the reportermoiety of the conjugate.

FIG. 7D. The primer is deprotected by cleavage of the linker, releasingthe polymerase and reporter.

FIG. 7E. The deprotection reaction is optionally confirmed by lack ofdetection of the reporter moiety.

FIGS. 8A-8B Demonstration of elongation of a primer bypolymerase-nucleotide conjugates with varying numbers of tetherednucleotides on SDS-PAGE.

FIG. 8A. Elongation of the primer by wild-type TdT (with up to fivetethered nucleotides) and a TdT mutant with only one tetherednucleotide.

FIG. 8B. Elongation of a primer by conjugates of TdT mutants withvarious attachment points for the tethered nucleotide.

FIGS. 9A-9B. Demonstration of the DNA synthesis reaction cycle bySDS-PAGE and capillary electrophoresis (CE).

FIG. 9A. SDS-PAGE analysis of protein-DNA complex formation anddissociation upon elongation of a primer by a polymerase-nucleotideconjugate and cleavage of the linker.

FIG. 9B. Capillary electropherograms of reaction products from FIG. 9A.

FIG. 10 . Capillary electropherograms of reaction time courses for theextension of a 25 nM DNA primer by 16 μM TdT-dATP, -dCTP, -dGTP, and-dTTP conjugates, followed by photolysis.

FIGS. 11A-11B. Demonstration of synthesis of a 4-mer (5′-CTAG-3′).

FIG. 11A. Procedure for synthesis and sequence-verification of extensionproducts. Step 2: SEQ ID NO:15, Step 3: Top to bottom: SEQ ID NO:16, SEQID NO:23.

FIG. 11B. Sequencing electropherogram of one of the clones (SEQ IDNO:17).

FIG. 12 . Demonstration of free nucleotide incorporation into a primerthat is tethered to a polymerase via its incorporated tetherednucleotide, analyzed by CE. FIGS. 13A-13C. Experimental setup todemonstrate that scarred DNA can serve as a template for accuratecomplementary DNA synthesis.

FIG. 13A. Scheme for synthesizing a polynucleotide consisting ofnucleotides with a 3-acetamidopropynyl modification (“scars”)

FIG. 13B. Capillary electrophoresis analysis of the synthesized“scarred” polynucleotide.

FIG. 13C. qPCR amplification of the “scarred” polynucleotide.

FIGS. 14A-14B. Demonstration of synthesis of a 10-mer (5′-CTACTGACTG-3′)(SEQ ID NO:18).

FIG. 14A. Procedure for synthesis and sequence-verification of extensionproducts. Step 1: SEQ ID NO: 19, Step 2: SEQ ID NO: 20, Step 3: Top tobottom: SEQ ID NO: 21, SEQ ID NO: 24.

FIG. 14B. Sequencing electropherogram of one of the clones (SEQ ID NO:22) and analysis of the synthesis steps.

DETAILED DESCRIPTION

Provided herein is a conjugate comprising a polymerase and a nucleosidetriphosphate, wherein the polymerase and the nucleoside triphosphate arelinked via a linker that comprises a cleavable linkage. An example ofsuch a conjugate is shown in FIG. 3C and FIG. 4C. The polymerase moietyof a conjugate can elongate a nucleic acid using its linked nucleosidetriphosphate (i.e., the polymerase can catalyze the attachment of anucleotide to which it is joined onto a nucleic acid) and remainsattached to the elongated nucleic acid via the linker until the linkeris cleaved.

In some embodiments, once the polymerase of a conjugate has incorporatedits tethered nucleotide into a nucleic acid, further elongations of thatnucleic acid by other polymerase-nucleotide conjugates are hindered viaan effect referred to herein as “shielding”, where the term “shielding”refers to a phenomenon in which 1) the attached polymerase moleculehinders other conjugate molecules from accessing the 3′ OH of theelongated DNA molecule and 2), the nucleoside triphosphate moleculestethered to other conjugate molecules are hindered from accessing thecatalytic site of the polymerase that has become attached to the end ofthe elongated nucleic acid. In some embodiments, further elongation ofthe nucleic acid may be terminated without the need for additionalblocking groups on the tethered nucleoside triphosphate. The terminationof elongation caused by the shielding effect may be reversed by cleavageof the linker, which releases the tethered polymerase and therebyreveals the 3′ end of the elongated nucleic acid to enable subsequentelongation by another conjugate.

In any embodiment, conjugates may comprise additional moieties thatcontribute to termination of elongation of a nucleic acid once thetethered nucleotide has been incorporated. For example, 3′ O-modified orbase-modified reversible terminator deoxynucleoside triphosphates(RTdNTPs) that are well known and reviewed in a variety of publications,including Chen, Fei, et al. (Genomics, proteomics & bioinformatics 11.1(2013): 34-40.), may be tethered to the polymerase. Reversibleterminator nucleotide refers to a chemically modified nucleosidetriphosphate analog that can be incubated in solution with a polymeraseand a nucleic acid and, once incorporated into a nucleic acid molecule,hinders further elongation in the reaction. When a conjugate comprisinga polymerase and an RTdNTP is used for the extension of a nucleic acid,besides cleavage of the linker, also deprotection of the RTdNTP may berequired to enable an extended nucleic acid to undergo furthernucleotide addition.

In some embodiments, the conjugate may be fluorescent, which may beuseful in sequencing applications. In some embodiments, the nucleosidetriphosphate may be linked to a cysteine residue in the polymerase.However, other chemistries may be used to link proteins and nucleosidetriphosphate and, as such, in some cases, the nucleoside triphosphatemay be linked to a non-cysteine residue in the polymerase.

The cleavable linker should be capable of being selectively cleavedusing a stimulus (e.g., light, a change in its environment or exposureto a chemical or enzyme) without breakage of other bonds in the nucleicacid. In some embodiments, the cleavable linkage may be a disulfidebond, which can be readily broken using a reducing agent (e.g.,β-mercaptoethanol or the like). Cleavable bonds that may be suitable mayinclude, but are not limited to, the following: base-cleavable sitessuch as esters, particularly succinates (cleavable by, for example,ammonia or trimethylamine), quaternary ammonium salts (cleavable by, forexample, diisopropylamine) and urethanes (cleavable by aqueous sodiumhydroxide); acid-cleavable sites such as benzyl alcohol derivatives(cleavable using trifluoroacetic acid), teicoplanin aglycone (cleavableby trifluoroacetic acid followed by base), acetals and thioacetals (alsocleavable by trifluoroacetic acid), thioethers (cleavable, for example,by HF or cresol) and sulfonyls (cleavable by trifluoromethane sulfonicacid, trifluoroacetic acid, thioanisole, or the like);nucleophile-cleavable sites such as phthalamide (cleavable bysubstituted hydrazines), esters (cleavable by, for example, aluminumtrichloride); and Weinreb amide (cleavable by lithium aluminum hydride);and other types of chemically cleavable sites, includingphosphorothioate (cleavable by silver or mercuric ions),diisopropyldialkoxysilyl (cleavable by fluoride ions), diols (cleavableby sodium periodate), and azobenzenes (cleavable by sodium dithionite).Other cleavable bonds will be apparent to those skilled in the art orare described in the pertinent literature and texts (e.g., Brown (1997)Contemporary Organic Synthesis 4(3); 216-237).

In particular embodiments, a photocleavable (“PC”) linker (e.g., auv-cleavable linker) may be employed. Suitable photocleavable linkersfor use may include ortho-nitrobenzyl-based linkers, phenacyl linkers,alkoxybenzoin linkers, chromium arene complex linkers, NpSSMpact linkersand pivaloylglycol linkers, as described in Guillier et al (Chem Rev.2000 Jun. 14; 100(6):2091-158). Exemplary linking groups that may beemployed in the subject methods may be described in Guillier et al,supra and Olejnik et al (Methods in Enzymology 1998 291:135-154), andfurther described in U.S. Pat. No. 6,027,890; Olejnik et al (Proc. Natl.Acad Sci, 92:7590-94); Ogata et al. (Anal. Chem. 2002 74:4702-4708); Baiet al (Nucl. Acids Res. 2004 32:535-541); Zhao et al (Anal. Chem. 200274:4259-4268); and Sanford et al (Chem Mater. 1998 10:1510-20), and arepurchasable from Ambergen (Boston, Mass.; NHS-PC-LC-Biotin), LinkTechnologies (Bellshill, Scotland), Fisher Scientific (Pittsburgh, Pa.)and Calbiochem-Novabiochem Corp. (La Jolla, Calif.).

In other embodiments, the linkage may be cleaved by an enzyme. Forexample, an amide linkage may be cleaved by a protease, an ester linkagemay be cleaved by an esterase, and a glycosidic linkage may be cleavedby a glycosylase. In some embodiments, the cleavage reagent may alsobreak bonds in the attached polymerase, e.g. a protease may also digestthe polymerase.

In a conjugate, the linker is considered to be at least the atoms thatconnect the base, the sugar, or the α-phosphate of a nucleotide to aC_(α) atom in the backbone of the polymerase. In some embodiments, thepolymerase and the nucleotide are covalently linked and the distancebetween the linked atom of the nucleotide and the C_(α) atom in thebackbone of the polymerase to which it is attached may be in the rangeof 4-100 Å, e.g., 15-40 or 20-30 Å, although this distance may varydepending on where the nucleoside triphosphate is tethered. In someembodiments, the linker may be a PEG or polypeptide linker, although,again, there is considerable flexibility on the type of linker used. Insome embodiments, the linker should be joined to the base of thenucleotide at an atom that is not involved in base pairing. In suchembodiments, the linker is considered to be at least the atoms thatconnect a C_(α) atom in the backbone of the polymerase to any atom inthe monocyclic or polycyclic ring system bonded to the 1′ position ofthe sugar (e.g. pyrimidine or purine or 7-deazapurine or8-aza-7-deazapurine). For example, in the conjugate depicted in FIG. 4D,the linker is joined to the carbon atom at the 5 position of thecytosine nucleobase and to the C_(α) atom of the cysteine residue of thepolymerase. In other embodiments, the linker should be joined to thebase of the nucleotide at an atom that is involved in base pairing. Inother embodiments, the linker should be joined to the sugar or to theα-phosphate of the nucleotide.

In all embodiments, the linker used should be sufficiently long to allowthe nucleoside triphosphate to access the active site of the polymeraseto which it is tethered. As will be described in greater detail below,the polymerase of a conjugate is capable of catalyzing the addition ofthe nucleotide to which it is linked onto the 3′ end of a nucleic acid.

A nucleic acid may be at least 3 nucleotides in length, at least 10nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least500 nucleotides, at least 1,000 nucleotides or at least 5,000nucleotides in length and can be fully single-stranded or at leastpartially double-stranded, e.g., hybridized to another molecule (i.e.part of a duplex) or to itself (e.g., in the form of a hairpin). In anyembodiment, a nucleic acid can be an oligonucleotide, which may be atleast 3 nucleotides in length, e.g., at least 10 nucleotides, at least50 nucleotides, at least 100 nucleotides in length, at least 500nucleotides up to 1,000 nucleotides or more in length and can be fullysingle-stranded or at least partially double-stranded, e.g., hybridizedto another molecule (i.e. part of a duplex) or to itself (e.g., in theform of a hairpin). In some embodiments, an oligonucleotide may behybridized to a template nucleic acid. In these embodiments, thetemplate nucleic acid may be at least 20 nucleotides in length, e.g., atleast 80 nucleotides in length, at least 150 nucleotides in length, atleast 300 nucleotides in length, at least 500 nucleotides in length, atleast 2000 nucleotides in length, at least 4000 nucleotides in length orat least 10,000 nucleotides. In some cases, a nucleic acid can be partof a natural DNA substrate, e.g. it may be a strand of a plasmid. If anucleic acid is double stranded, it can have a 3′ overhang.

Also provided herein is a set of the conjugates summarized above,wherein the conjugates correspond to (i.e., have a base-pairingcapability that is the same as) G, A, T (or U) and C (i.e.,deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP),deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP)).

In some embodiments, these conjugates are in separate containers. Inother embodiments, the conjugates may be in the same container,particularly if they are to be used for sequencing. The nucleotides usedherein may contain adenine, cytosine, guanine, and thymine bases, and/orbases that base pair with a complementary nucleotide and are capable ofbeing used as a template by a DNA or RNA polymerase, e.g.,7-deaza-7-propargylamino-adenine, 5-propargylamino-cytosine,7-deaza-7-propargylamino-guanosine, 5-propargylamino-uridine,7-deaza-7-hydroxymethyl-adenine, 5-hydroxymethyl-cytosine,7-deaza-7-hydroxymethyl-guanosine, 5-hydroxymethyl-uridine,7-deaza-adenine, 7-deaza-guanine, adenine, guanine, cytosine, thymine,uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza-guanosine,2-thio-adenine, 2-thio-7-deaza-adenine, isoguanine, 7-deaza-guanine,5,6-dihydrouridine, 5,6-dihydrothymine, xanthine, 7-deaza-xanthine,hypoxanthine, 7-deaza-xanthine, 2,6 diamino-7-deaza purine,5-methyl-cytosine, 5-propynyl-uridine, 5-propynyl-cytidine,2-thio-thymine or 2-thio-uridine are examples of such bases, althoughothers are known. An exemplary set of conjugates for synthesizing and/orsequencing a DNA molecule may include a DNA polymerase linked to adeoxyribonucleotide triphosphate selected from deoxyriboadenosinetriphosphate (dATP), deoxyriboguanosine triphosphate (dGTP),deoxyribocytidine triphosphate (dCTP), deoxyribothymidine triphosphate(dTTP), and/or other deoxyribonucleotides that base pair in the same wayas those deoxyribonucleotides. An exemplary set of conjugates forsynthesizing an RNA molecule may include an RNA polymerase linked to aribonucleotide triphosphate selected from adenosine triphosphate (ATP),guanosine triphosphate (GTP), cytidine triphosphate (dCTP), and uridinetriphosphate (UTP), and/or other ribonucleotides that base pair in thesame way as those ribonucleotide triphosphates.

The above described conjugates can be used in a method of nucleic acidsynthesis. In some embodiments, this method may comprise: incubating anucleic acid with a first conjugate under conditions in which thepolymerase catalyzes the covalent addition of the nucleotide of thefirst conjugate onto the 3′ hydroxyl of the nucleic acid, to make anextension product. This reaction can be performed using a nucleic acidthat is attached to a solid support or that is in solution, i.e., nottethered to a solid support. After elongation of the nucleic acid by thefirst desired nucleotide, the method may comprise a deprotection stepwherein the cleavable linkage of the linker is cleaved, therebyreleasing the polymerase from the extension product. This may be done byexposing the reaction products to reducing conditions if the cleavablelinkage is a disulfide bond. However, other chemistries and reagents areavailable for this step. In some embodiments, the nucleosidetriphosphate may be a RTdNTP and the deprotection step of the methodfurther comprises removing the blocking group (i.e., removing theterminator group) from the added nucleotide to produce the deprotectedextension product. Deprotection enables subsequent extension of thenucleic acid, and thus allows these steps to be repeated cyclically toproduce an extension product of defined sequence. Specifically, in someembodiments, the method may further comprise, after deprotection:incubating the deprotected extension product with a second conjugateunder conditions in which polymerase catalyzes the covalent addition ofthe nucleotide of the second conjugate onto the 3′ end of the extensionproduct.

In some embodiments, the method may involve (a) incubating a nucleicacid with a first conjugate under conditions in which the polymerasecatalyzes the covalent addition of the nucleotide of the first conjugate(i.e., a single nucleotide) onto the 3′ hydroxyl of the nucleic acid, tomake an extension product; (b) cleaving the cleavable linkage of thelinker, thereby releasing the polymerase from the extension product anddeprotecting the extension product; (c) incubating the deprotectedextension product with a second conjugate of claim 1 under conditions inwhich the polymerase catalyzes the covalent addition of the nucleotideof the second conjugate onto the 3′ end of the extension product, tomake a second extension product; (d) repeating steps (b)-(c) on thesecond extension product multiple times (e.g., 2 to 100 or more times)to produce an extended oligonucleotide of a defined sequence. Steps(b)-(c) may be repeated as many times as necessary until an extensionproduct of a defined sequence and length is synthesized. The end productmay be 2-100 bases in length, although, in theory, the method can beused to produce products of any length, including greater than 200 basesor greater than 500 bases.

In certain embodiments, cleavage of the linker may leave a “scar” (i.e.,part of the linker) on each or some of the added nucleotides. In otherembodiments, cleavage of the linker does not produce a scar.

In some embodiments, scars may be further derivatized (e.g. byalkylation of thiol-containing scars using iodoacetamide) following eachdeprotection step. In other embodiments, all scars in the end productmay be simultaneously derivatized (e.g. by acetylation of propargylaminoscars using NHS acetate.)

In some embodiments, the product may be amplified, e.g., by PCR or someother method, to yield a product without scars (as demonstrated inExample 4).

A method of sequencing is also provided. These methods may compriseincubating a duplex comprising a primer and a template with acomposition comprising a set of conjugates, wherein the conjugatescorrespond to G, A, T and C and are distinguishably labeled, e.g.,fluorescently labeled; detecting which nucleotide has been added to theprimer by detecting a label that is tethered to the polymerase that hasadded the nucleotide to the primer; deprotecting the extension productby cleaving the linker; and repeating the incubation, detection anddeprotection steps to obtain the sequence of at least part of thetemplate.

Also provided is a reagent set that can be used to make a conjugatedescribed above. In some embodiments, this reagent set may comprise apolymerase that has been modified to contain a single cysteine on itssurface; and a set of nucleoside triphosphates, wherein each of thenucleoside triphosphates is linked to a sulfhydryl-reactive group. Insome embodiments, the nucleoside triphosphates correspond to G, A, T andC. As noted above, the nucleoside triphosphates may be reversibleterminators. In this reagent set, the nucleoside triphosphates maycomprise a linker that has a length in the range of 4-100 Å, e.g., 15-40Å or 20-30 Å.

In any embodiment, the polymerase can be a template-independentpolymerase, i.e., a terminal deoxynucleotidyl transferase or DNAnucleotidylexotransferase, which terms are used interchangeably to referto an enzyme having activity 2.7.7.31 using the IUBMB nomenclature. Adescription of such enzymes can be found in Bollum, F. J.Deoxynucleotide-polymerizing enzymes of calf thymus gland. V.Homogeneous terminal deoxynucleotidyl transferase. J. Biol. Chem. 246(1971) 909-916; Gottesman, M. E. and Canellakis, E. S. The terminalnucleotidyltransferases of calf thymus nuclei. J. Biol. Chem. 241 (1966)4339-4352; and Krakow, J. S., Coutsogeorgopoulos, C. and Canellakis, E.S. Studies on the incorporation of deoxyribonucleic acid. BiochimBiophys. Acta 55 (1962) 639-650, among others.

Terminal transferase embodiments may be useful for DNA synthesis.

In any embodiment, the polymerase can be a template-dependentpolymerase, i.e., a DNA-directed DNA polymerase (which terms are usedinterchangeably to refer to an enzyme having activity 2.7.7.7 using theIUBMB nomenclature), or an DNA-directed RNA polymerase. A description ofsuch enzymes can be found in Richardson, A. Enzymatic synthesis ofdeoxyribonucleic acid. XIV. Further purification and properties ofdeoxyribonucleic acid polymerase of Escherichia coli. J. Biol. Chem. 239(1964) 222-232; Schachman, A. Enzymatic synthesis of deoxyribonucleicacid. VII. Synthesis of a polymer of deoxyadenylate anddeoxythymidylate. J. Biol. Chem. 235 (1960) 3242-3249; and Zimmerman, B.K. Purification and properties of deoxyribonucleic acid polymerase fromMicrococcus lysodeikticus. J. Biol. Chem. 241 (1966) 2035-2041.

In any of the above-summarized embodiments, the nucleoside triphosphatemay be a deoxyribonucleoside triphosphate or a ribonucleosidetriphosphate. In some embodiments, a conjugate may comprise an RNApolymerase linked to a ribonucleoside triphosphate. In theseembodiments, the nucleotide added to the nucleic acid may be aribonucleotide. In other embodiments, a conjugate comprises an DNApolymerase linked to a deoxyribonucleoside triphosphate. In theseembodiments, the nucleotide added to the nucleic acid may be adeoxyribonucleotide.

In any embodiment, the polymerase used may have an amino acid sequencethat is at least 80% identical to, e.g., at least 90% or at least 95%identical to a wild type polymerase.

In some embodiments, the yield per nucleotide addition step may be atleast 70%, at least 80%, at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99%, for example 91% or 99.5%. The yield per stepof any implementation of the method may be increased by optimizing theconditions. As would be recognized, a nucleic acid manufactured by thepresented method may be purified, e.g., by liquid chromatography, priorto use.

In any embodiment, the conjugate may additionally comprise additionalpolypeptide domains fused to the polymerase. For example, maltosebinding protein may be fused to the N-terminus of Terminaldeoxynucleotidyl transferase to enhance its solubility and/or to enableamylose affinity purification. In any embodiment, the nucleosidetriphosphate may be a reversible terminator.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Further details of the reagents and methods described above may be foundbelow. Some of this description relates to the TdT. The principle ofthis description can be applied to other template-independentpolymerases and template-dependent polymerases, too.

Tethered Nucleotides can have a High Effective Concentration, EnablingFast Incorporation Kinetics.

A tethered nucleotide will have a certain occupancy rate with the activesite of the polymerase depending on the length and geometry of thelinker and its attachment site on the protein. This rate can beexpressed as an effective concentration (the concentration of freenucleotide that would give an equivalent occupancy rate). By varying thelinker properties and attachment site, it is possible to control theeffective concentration of the nucleotide, enabling high effectiveconcentrations and therefore fast incorporations. For example, a veryrough calculation suggests that the effective concentration of a dNTPtethered by a 20 Å linker will be ˜50 mM, (one molecule in the volume ofa sphere with 20 Å radius). In this example, one could increase thelocal concentration of the dNTP by shortening the linker, or decrease itby lengthening the linker.

Attachment Position of the Linker on the Polymerase

In some embodiments, the linker is specifically attached to an aminoacid of the polymerase (see FIG. 2 for a schematic drawing). In thesecases, it is preferable to attach the linker to an amino acid at aposition that can be mutated without loss of the polymerase activity,e.g. positions 180, 188, 253 or 302 of murine TdT (numbering as in thecrystal structure PDB ID: 4127). It is preferable to not attach thelinker to an amino acid involved in the catalytic activity of thepolymerase to avoid interfering with catalysis. Residues known to beinvolved with catalysis and methods for determining if a residue isinvolved with catalysis (e.g. by site-specific mutagenesis) will beapparent to those skilled in the art and are reviewed in literature(e.g. Joyce et al. (Journal of Bacteriology 177.22 (1995): 6321.) andJara and Martinez (The Journal of Physical Chemistry B 120.27 (2016):6504-6514.))

Length of the Linker

In any embodiment, the length of the linker may be longer than thedistance between the attachment position of the linker on the polymeraseand the attachment position on the nucleoside triphosphate when it isbound to the catalytic site. In some cases, steric restrictions, e.g.due to the polymerase or due to the linker can restrict mobility of thetethered nucleoside triphosphate requiring an increased linker length toenable the tethered nucleoside triphosphate to access the catalytic siteof the polymerase in a productive conformation. For example, the linkerlength may exceed the distance between its two attachment points by 2-3Å or 5-10 Å, or 10-25 Å, or longer.

Strategies for Site-Specific Attachment of a Linker to a Polymerase.

In some embodiments, the tethered nucleoside triphosphate may bespecifically attached to a cysteine residue of the polymerase using asulfhydryl-specific attachment chemistry. Possible sulfhydryl specificattachment chemistries include, but are not limited to ortho-pyridyldisulfide (OPSS) (as exemplified in FIG. 3 and demonstrated in Example1), maleimide functionalities (as exemplified in FIG. 4 and demonstratedin Example 2), 3-arylpropiolonitrile functionalities, allenamidefunctionalities, haloacetyl functionalities such as iodoacetyl orbromoacetyl, alkyl halides or perfluroaryl groups that can favorablyreact with sulfhydryls surrounded by a specific amino acid sequence(Zhang, Chi, et al. Nature chemistry 8, (2015) 120-128.). Otherattachment chemistries for specific labeling of cysteine residues willbe apparent to those skilled in the art or are described in thepertinent literature and texts (e.g., Kim, Younggyu, et al, Bioconjugatechemistry 19.3 (2008): 786-791.).

In other embodiments, the linker could be attached to a lysine residuevia an amine-reactive functionality (e.g. NHS esters, Sulfo-NHS esters,tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides,etc.).

In other embodiments, the linker may be attached to the polymerase viaattachment to a genetically inserted unnatural amino acid, e.g.p-propargyloxyphenylalanine or p-azidophenylalanine that could undergoazide-alkyne Huisgen cycloaddition, though many suitable unnatural aminoacids suitable for site-specific labeling exist and can be found in theliterature (e.g. as described in Lang and Chin., Chemical reviews 114.9(2014): 4764-4806.).

In other embodiments, the linker may be specifically attached to thepolymerase N-terminus. In some embodiments, the polymerase is mutated tohave an N-terminal serine or threonine residue, which may bespecifically oxidized to generate an N-terminal aldehyde for subsequentcoupling to e.g. a hydrazide. In other embodiments, the polymerase ismutated to have an N-terminal cysteine residue that can be specificallylabeled with an aldehyde to form a thiazolidine. In other embodiments,an N-terminal cysteine residue can be labeled with a peptide linker viaNative Chemical Ligation.

In other embodiments, a peptide tag sequence may be inserted into thepolymerase that can be specifically labeled with a synthetic group by anenzyme, e.g. as demonstrated in the literature using biotin ligase,transglutaminase, lipoic acid ligase, bacterial sortase andphosphopantetheinyl transferase (e.g. as described in refs. 74-78 ofStephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

In other embodiments, the linker is attached to a labeling domain fusedto the polymerase. For example, a linker with a corresponding reactivemoiety may be used to covalently label SNAP tags, CLIP tags, HaloTagsand acyl carrier protein domains (e.g. as described in refs. 79-82 ofStephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

In other embodiments, the linker is attached to an aldehyde specificallygenerated within the polymerase, as described in Carrico et al. (Nat.Chem. Biol. 3, (2007) 321-322). For example, after insertion of an aminoacid sequence that is recognized by the enzyme formylglycine-generatingenzyme (FGE) into the polymerase, it may be exposed to FGE, which willspecifically convert a cysteine residue in the recognition sequence toformylglycine (i.e. producing an aldehyde). This aldehyde may then bespecifically labeled with e.g. a hydrazide or aminooxy moiety of alinker.

In some embodiments, a linker may be attached to the polymerase vianon-covalent binding of a moiety of the linker to a moiety fused to thepolymerase. Examples of such attachment strategies include fusing apolymerase to streptavidin that can bind a biotin moiety of a linker, orfusing a polymerase to anti-digoxigenin that can bind a digoxigeninmoiety of a linker.

In some embodiments, site-specific labeling may lead to an attachment ofthe linker to the polymerase that may readily be reversed (e.g. anortho-pyridyl disulfide (OPSS) group that forms a disulfide bond with acysteine that can be cleaved using reducing agents, e.g. using TCEP),other attachment chemistries will produce permanent attachments.

In any embodiment, the polymerase may be mutated to ensure specificattachment of the tethered nucleotide to a particular location of thepolymerase, as will be apparent to those skilled in the art. Forexample, with sulfhydryl-specific attachment chemistries such asmaleimides or ortho-pyridyl disulfides, accessible cysteine residues inthe wild-type polymerase may be mutated to a non-cysteine residue toprevent labeling at those positions. On this “reactive cysteine-free”background, a cysteine residue may be introduced by mutation at thedesired attachment position. These mutations preferentially do notinterfere with the activity of the polymerase.

Other strategies for site-specific attachment of synthetic groups toproteins will be apparent to those skilled in the art and are reviewedin literature, (e.g. Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011)876-884).

Strategies for Attaching a Linker to a Nucleoside Triphosphate.

In some embodiments, the linker is attached the 5 position ofpyrimidines or the 7 position of 7-deazapurines. In other embodiments,the linker may be attached to an exocyclic amine of a nucleobase, e.g.by N-alkylating the exocyclic amine of cytosine with a nitrobenzylmoiety as discussed below. In other embodiments, the linker may beattached to any other atom in the nucleobase, sugar, or α-phosphate, aswill be apparent to those skilled in the art.

Certain polymerases have a high tolerance for modification of certainparts of a nucleotide, e.g. modifications of the 5 position ofpyrimidines and the 7 position of purines are well-tolerated by somepolymerases (He and Seela., Nucleic Acids Research 30.24 (2002):5485-5496.; or Hottin et al., Chemistry. 2017 Feb. 10; 23(9):2109-2118).In some embodiments, the linker is attached to these positions.

In some examples, a polymerase-nucleotide conjugate is prepared by firstsynthesizing an intermediate compound comprising a linker and anucleoside triphosphate (referred to herein as a “linker-nucleotide”),and then this intermediate compound is attached to the polymerase. Insome examples, nucleosides with substitutions compared to naturalnucleosides, e.g. pyrimidines with 5-hydroxymethyl or 5-propargylaminosubstituents, or 7-deazapurines with 7-hydroxymethyl or 7-propargylaminosubstituents may be useful starting materials for preparinglinker-nucleotides. An exemplary set of nucleosides with 5- and7-hydroxymethyl substituents that may be useful for preparinglinker-nucleotides is shown below:

An exemplary set of nucleosides with 5- and 7-deaza-7-propargylaminosubstituents that may be useful for preparing linker-nucleotides isshown below:

These nucleosides are also commercially available as deoxyribonucleosidetriphosphates.

In Example 2, linker-nucleotides comprising a3-(((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group attached to the 5position of pyrimidines and the 7 position of 7-deazapurines wereprepared by reacting nucleoside triphosphates containing 5- and7-propargylamino substituents with a precursor molecule comprising anitrobenzyl NHS carbonate ester (as shown in FIG. 4 ).

Linker Cleavage Strategies

As described above, the linker may be attached to various positions onthe nucleotide, and a variety of cleavage strategies may be used. Thosestrategies may include, but are not limited to, the following examples:

In some embodiments, the linker may be cleaved by exposure to a reducingagent such as dithiothreitol (DTT). For example, a linker comprising a4-(disulfaneyl)butanoyloxy-methyl group attached to the 5 position of apyrimidine or the 7 position of a 7-deazapurine may be cleaved byreducing agents (e.g. DTT) to produce a 4-mercaptobutanoyloxymethyl scaron the nucleobase. This scar may undergo intramolecularthiolactonization to eliminate a 2-oxothiolane, leaving a smallerhydroxymethyl scar on the nucleobase. An example of such a linkerattached to the 5 position of cytosine is depicted below, but thestrategy is applicable to any suitable nucleobase:

In other embodiments, the linker may be cleaved by exposure to light.For example a linker comprising (2-nitrobenzyl)oxymethyl group may becleaved with 365 nm light, leaving a hydroxymethyl scar, e.g. asdepicted for cytosine below, but as is applicable to any suitablenucleobase:

(where, e.g., R″═H or R″═CH3 or R″=t-Bu.)

In other embodiments, the linker may comprise a3-(((2-nitrobenzyl)oxy)carbonyl)aminopropynyl group that may be cleavedwith 365 nm light to release a nucleobase with a propargylamino scar.This strategy was used in Example 2 and is depicted for cytosine below,but is applicable to any suitable nucleobase:

In other embodiments, the linker may comprise an acyloxymethyl groupthat may be cleaved with a suitable esterase to release a nucleobasewith a hydroxymethyl scar, e.g. as depicted for cytosine below, but asis applicable to any suitable nucleobase:

In such embodiments, the linker may comprise additional atoms (includedin R′ above) adjacent to the ester that increase the activity of theesterase towards the ester bond.

In other embodiments, the linker may comprise an N-acyl-aminopropynylgroup that may be cleaved with a peptidase to release a nucleobase withpropargylamino scar, e.g. as depicted for 5-propargylamino cytosinebelow, but as is applicable to any suitable nucleobase:

In such embodiments, the linker may comprise additional atoms (includedin R′ above) adjacent to the amide that increase the activity of thepeptidase towards the amide bond. In some embodiments, R′ is a peptideor polypeptide.

Cleavage of Peptide Bonds to Detach the Tethered Nucleotide

In some embodiments, one or more amino acids are inserted into thepolymerase that can serve as part of the cleavable linker for specificattachment of the nucleotide. In this case, the linker comprises theinserted amino acids, and the cleavable linkage is considered to be oneor more bonds of the inserted amino acid(s). For example, peptide bondsmay be cleaved using a peptidase (which terms are used interchangeablyto refer to an enzyme having activity 3.4. using the IUBMB nomenclature)such as Proteinase K (EC 3.4.21.64 using the IUBMB nomenclature).

In some embodiments, the protein itself can be cleaved to detach thetethered nucleoside triphosphate from the polymerase. For example,peptide bonds before and/or after the attachment position of the linkermay be cleaved using a peptidase.

In some embodiments, amino acid positions close to the attachment pointof the linker may be mutated to ensure that peptide sequences near theattachment point are good substrates for the protease, as will beapparent to those skilled in the art. E.g., mutations into aliphaticamino acids as leucine or phenylalanine may be introduced to achievefast cleavage with proteinase K.

RTdNTP-Polymerase Conjugates.

In certain embodiments, nucleotide analogs are tethered that, whenfreely available in solution, do not terminate DNA synthesis uponincorporation. However, in other embodiments nucleotide analogscontaining a reversible terminator group, such as an O-azidomethyl orO—NH₂ group on the 3′ position of the sugar or an(alpha-tertbutyl-2-nitrobenzyl)oxymethl group on the 5 position ofpyrimidines or the 7 position of 7-deazapurines (for an overview see,e.g. Chen et al., Genomics, Proteomics & Bioinformatics 2013 11: 34-40.)are tethered. In these embodiments, the nucleotide analog prevents orhinders further elongation once incorporated into a nucleic acid andthus contributes to the conjugate's ability to achieve termination,possibly in addition to other properties of the conjugate thatcontribute to termination (e.g. shielding). In the case thatRTdNTP-polymerase conjugates do not rely on the shielding effect toachieve termination, e.g. when a 3′ modified RTdNTP is tethered to thepolymerase, the linker used may exceed 100 Å or 200 Å in length.

Shielding Effect by Polymerase-Nucleotide Conjugates

When a conjugate comprising a polymerase and a nucleoside triphosphateis incubated with a nucleic acid, it preferentially elongates thenucleic acid using its tethered nucleotide (as opposed to using thenucleotide of another conjugate molecule). As described above, thepolymerase then remains attached to the nucleic acid via its tether tothe added nucleotide (e.g. FIG. 3D and FIG. 4D) until exposed to somestimulus that causes cleavage of the linkage to the added nucleotide. Inthis situation, further extensions by polymerase-nucleotide conjugatesare hindered due to “shielding” when: 1) the attached polymerasemolecule hinders other conjugates from accessing the 3′ OH of theextended DNA molecule and 2), other nucleoside triphosphates in thesystem are hindered from accessing the catalytic site of the polymerasethat remains attached to the 3′ end of the extended nucleic acid. (Theextent of shielding may be described as the extent to which both ofthese interactions are hindered.) To enable subsequent extensions, thelinker tethering the incorporated nucleotide to the polymerase can becleaved, releasing the polymerase from the nucleic acid and thereforere-exposing its 3′ OH group for subsequent elongation (e.g. as depictedin FIG. 3E and FIG. 4E).

Methods for nucleic acid synthesis and sequencing provided herein thatemploy the shielding effect to achieve termination comprise an extensionstep wherein a nucleic acid is exposed to conjugates preferentially inthe absence of free (i.e. untethered) nucleoside triphosphates, becausethe termination mechanism of shielding may not prevent theirincorporation into the nucleic acid. As shown in Example 3, exposing aprimer that has been extended by a TdT-dCTP conjugate to free dCTPresults in several additional elongations.

In some embodiments, termination of further elongation may be“complete”, meaning that after a nucleic acid molecule has beenelongated by a conjugate, further elongations cannot occur during thereaction. In other embodiments, termination of further elongation may be“incomplete”, meaning that further elongations can occur during thereaction but at a substantially decreased rate compared to the initialelongation, e.g. 100 times slower, or 1000 times slower, or 10,000 timesslower, or more. Conjugates that achieve incomplete termination maystill be used to extend a nucleic acid by predominantly a singlenucleotide (e.g. in methods for nucleic acid synthesis and sequencing)when the reaction is stopped after an appropriate amount of time.

In some embodiments, the reagent containing the conjugate mayadditionally contain polymerases without tethered nucleosidetriphosphates, but those polymerases should not significantly affect thereaction because there are no free dNTPs in the mix.

Reagents based on conjugates employing the shielding effect to achievetermination preferentially only contain polymerase-nucleotide conjugatesin which all polymerases remain folded in the active conformation. Insome cases, if the polymerase moiety of a conjugate is unfolded, itstethered nucleoside triphosphate may become more accessible to thepolymerase moieties of other conjugate molecules. In these cases, theunshielded nucleotides may be more readily incorporated by otherconjugate molecules, circumventing the termination mechanism.

Polymerase-nucleotide conjugates employing the shielding effect toachieve termination are preferentially only labeled with a singlenucleoside triphosphate moiety. Polymerase-nucleotide conjugates labeledwith multiple nucleoside triphosphates that can access the catalyticsite can, in some cases, incorporate multiple nucleoside triphosphatesinto the same nucleic acid (e.g. as demonstrated with the conjugate ofwt TdT labeled with up to 5 nucleoside triphosphates in Example 1).Additional tethered nucleotides may therefore lead to additional,undesired nucleotide incorporations into a nucleic acid during areaction. Furthermore, only one tethered nucleoside triphosphates canoccupy the (buried) catalytic site of its polymerase at a time so theother tethered nucleoside triphosphate(s) may have an increasingaccessibility to the polymerase moieties of other conjugate molecules,as discussed below. Strategies for site-specifically tethering at mostone nucleoside triphosphate to a polymerase are described above.

Polymerase-nucleotide conjugates employing the shielding effect toachieve termination preferentially comprise as short of a linker aspossible that still enables the nucleoside triphosphate to frequentlyaccess the catalytic site of its tethered polymerase molecule in aproductive conformation, in order to enable fast incorporation of thenucleotide into a nucleic acid. Such conjugates may also preferentiallyemploy an attachment position of the linker to the polymerase as closeto the catalytic site as possible, enabling use of a shorter linker. Thelength of the linker will determine the maximum distance from theattachment point a tethered nucleoside triphosphate or a tetherednucleic acid can reach. A smaller distance may lead to a reducedaccessibility of the tethered moiety to other polymerase-nucleotidemolecules, as discussed below. Linkers used in Examples 1 and 2 areapproximately 24 and 28 Å long. Shorter linkers, e.g. with lengths of8-15 Å may increase shielding; longer linkers, e.g. linkers longer than50 Å, 70 Å or 100 Å, may reduce shielding.

The shielding effect may be influenced by a combination of factorsincluding, but not limited to, to the structure of the polymerase, thelength of the linker, the structure of the linker, the attachmentposition of the linker to the polymerase, the binding affinity of thenucleoside triphosphate to the catalytic site of the polymerase, thebinding affinity of the nucleic acid to the polymerase, the preferredconformation of the polymerase, and the preferred conformation of thelinker.

One contribution to shielding can be steric effects that block the 3′ OHof a nucleic acid that has been elongated by a conjugate from reachinginto the catalytic site of another conjugate's polymerase moiety. Stericeffects may also hinder a tethered nucleoside triphosphate from reachinginto the catalytic site of another polymerase-nucleotide conjugatemolecule due to clashes between the conjugates that would occur duringsuch approaches. These steric effects may result in complete terminationif they completely block productive interactions between the tetherednucleoside triphosphate (or elongated nucleic acid) of one conjugatemolecule with another conjugate molecule, or may result in incompletetermination if they only hinder such intermolecular interactions.

Another contribution to shielding arises from the binding affinity ofthe tethered nucleoside triphosphate to the catalytic site of thepolymerase. The tethered nucleoside triphosphate of a conjugate willhave a high effective concentration with respect to the catalytic siteof its tethered polymerase so it may remain bound to that site much ofthe time. When the nucleoside triphosphate is bound to the catalyticsite of its tethered polymerase molecule it is unavailable forincorporation by other polymerase molecules. Thus, tethering reduces theeffective concentration of nucleoside triphosphates available forintermolecular incorporation (i.e. incorporation catalyzed by apolymerase molecule to which the nucleotide is not tethered). Thisshielding effect can enhance termination by reducing the rate by which anucleic acid is elongated using the nucleoside triphosphate moiety ofone conjugate molecule by the polymerase moiety of another conjugatemolecule.

Another contribution to shielding arises from the binding affinity ofthe 3′ region of a nucleic acid molecule to the catalytic site of apolymerase molecule. After elongation by a conjugate, the nucleic acidis tethered to the conjugate via it's 3′ terminal nucleotide and willhave a high effective concentration with respect to the catalytic siteof its tethered polymerase so it may remain bound to that site much ofthe time. When the nucleic acid is bound to the catalytic site of itstethered polymerase molecule it is unavailable for elongation by otherconjugate molecules. This effect can enhance termination by reducing therate by which a nucleic acid that has been elongated by a firstconjugate is further elongated by other conjugate molecules.

Addition of Elements with Steric Restrictions to Increase the ShieldingEffect

In some embodiments, the polymerase-nucleotide conjugates compriseadditional moieties that sterically hinder the tethered nucleosidetriphosphate (or a tethered nucleic acid post-elongation) fromapproaching the catalytic sites of another conjugate molecule. Suchmoieties include polypeptides or protein domains that can be insertedinto a loop of the polymerase, and those and other bulky molecules suchas polymers that can be site-specifically ligated e.g. to an insertedunnatural amino acid or specific polypeptide tag.

RTdNTP Termination Mechanisms in Combination with the Shielding Effect.

As described above, in some embodiments, a conjugate may comprise apolymerase and a tethered reversible terminator nucleoside triphosphate.Some RTdNTPs (particularly 3′ O-unblocked RTdNTPs) achieve incompletetermination when used freely in solution. A polymerase-nucleotideconjugate of such an RTdNTP that also employs a shielding effect mayachieve more complete termination than the RTdNTP used by itself. Insome embodiments, an RTdNTP with an(alpha-tertbutyl-2-nitrobenzyl)oxymethyl group attached to the 5position of a pyrimidine or the 7 position of a 7-deazapurine in anucleoside triphosphate e.g. described in Gardner et al. (Nucleic AcidsRes. 2012 August; 40(15):7404-1); or Stupi et al. (Angewandte ChemieInternational Edition 51.7 (2012): 1724-1727) may be employed. In someembodiments, the linker is attached to an atom in the terminating moietyof the RTdNTP. In other embodiments the linker is attached to an atom ofthe RTdNTP not in the terminating moiety.

Effective Concentration of Nucleoside Triphosphates Tethered to OtherPolymerase-Nucleotide Conjugates

As described earlier, a tethered nucleoside triphosphate has a higheffective concentration with respect to the catalytic site of itsattached polymerase moiety, enabling fast incorporation. The samenucleoside triphosphate has a much lower concentration with respect tothe catalytic site of other polymerase-moieties, leading to a slowerintermolecular nucleotide incorporation rate if intermolecularincorporations are possible. The effective concentration of nucleosidetriphosphates tethered to other conjugates is at most the absoluteconcentration of conjugates since each conjugate molecule comprises asingle nucleotide. Due to shielding effects that hinder accessibility ofthese nucleoside triphosphates, the effective concentration is furtherreduced.

Preventing an Elongated Nucleic Acid from Shifting in the PolymeraseCatalytic Site

An additional termination effect of polymerase-nucleotide conjugates canbe achieved by choosing a linker and attachment position that prevent anextended (and thus tethered) nucleic acid from shifting its tethered 3′end to the position where its 3′ OH can be activated to attack anincoming nucleoside triphosphate. This effect may be achieved if thetethered nucleoside triphosphate can access the nucleoside triphosphatebinding site of the polymerase but cannot reach a position where its 3′OH would correspond to the 3′ OH of an incoming nucleic acid.

Application of Polymerase-Nucleotide Conjugates to De Novo Nucleic AcidSynthesis

Described herein is a method for the de novo synthesis of nucleic acidsusing conjugates comprising a polymerase and a nucleoside triphosphate.In some embodiments of the method, conjugates comprise the polymeraseTerminal deoxynucleotidyl Transferase (TdT). In other embodiments, themethod may employ conjugates comprising another template-independentpolymerase or a template-dependent polymerase.

FIG. 1A illustrates a typical process for the stepwise synthesis of adefined sequence using a template-independent polymerase. A nucleic acidthat serves as an initial substrate for elongation (i.e. “startermolecule”) is incubated with a first polymerase-nucleotide conjugate.Once the nucleic acid has been elongated by the tethered nucleotide of aconjugate, no further elongations occur because the conjugates implementa termination mechanism, e.g. based on the shielding effect. In thesecond step of the process, the linker is cleaved to release thepolymerase and reverse the termination mechanism, thus enablingsubsequent elongations. The elongation products are then exposed to thesecond conjugate, and these two steps are iterated to elongate thenucleic acid by a defined sequence. FIG. 1B illustrates a synthesisprocedure using a conjugate comprising TdT and a photocleavable linkeras practiced in Example 2. As described above, other strategies areavailable for the attachment and cleavage of the linker.

For DNA synthesis applications, in particular template-independentpolymerases, i.e., a terminal deoxynucleotidyl transferase or DNAnucleotidylexotransferase, which terms are used interchangeably to referto an enzyme having activity 2.7.7.31 may be used. Polymerases with theability to extend single stranded nucleic acids include, but are notlimited to, Polymerase Theta (Kent et al., Elife 5 (2016): e13740.),polymerase mu (Juarez et al., Nucleic acids research 34.16 (2006):4572-4582.; or McElhinny et all., Molecular cell 19.3 (2005): 357-366.)or polymerases where template independent activity is induced, e.g. bythe insertion of elements of a template independent polymerase (Juarezet al., Nucleic acids research 34.16 (2006): 4572-4582). In other DNAsynthesis applications, the polymerase can be a template-dependentpolymerase i.e., a DNA-directed DNA polymerase (which terms are usedinterchangeably to refer to an enzyme having activity 2.7.7.7 using theIUBMB nomenclature).

For RNA synthesis applications, tethered ribonucleoside triphosphatesmay be used. In these embodiments, a RNA specific nucleotidyltransferase, such as E. coli Poly(A) Polymerase (IUBMB EC 2.7.7.19) orPoly(U) Polymerase, among others, may be employed. The RNA nucleotidyltransferases can contain modifications, e.g. single point mutations,that influence the substrate specificity towards a specific rNTP (Lundeet al., Nucleic acids research 40.19 (2012): 9815-9824.). In someembodiments, a very short tether between an RNA nucleotidyl transferaseand a ribonucleoside triphosphate may be used to induce a high effectiveconcentration of the nucleoside triphosphate, thereby forcingincorporation of an rNTP that might not be the natural substrate of thenucleotidyl transferase.

Initial Substrates for De Novo Nucleic Acid Synthesis.

Nucleic acid synthesis schemes using polymerase tethered nucleosidetriphosphates may require a nucleic acid substrate of at least 3-5 bases(or a molecule with similar properties) to initiate the synthesis. Thisinitial substrate can then be extended nucleotide-by-nucleotide into thedesired product. In some embodiments, the initial substrate may be anoligonucleotide primer synthesized using the phosphoramidite method (asdemonstrated in Example 1). In some cases, the particular sequence ofthe initiating primer may be used in downstream applications of thesynthesized nucleic acid. In some embodiments, the sequence of theinitial substrate may be removed from the synthesized nucleic acid aftersynthesis is complete, particularly if the initial substrate comprises acleavable linkage near it's 3′ terminus. For example, if the initialsubstrate is a primer that has a 3′ terminal deoxyuridine base, exposureof the elongated primer to USER Enzyme (i.e. a mixture of Uracil DNAglycosylase and Endonuclease VIII) will cleave the synthesized sequencefrom the initial substrate. However, other cleavable linkages may beused, e.g. a bridging phosphorothioate in the primer that could becleaved with silver or mercuric ions.

In some embodiments, a double-stranded DNA molecule may be employed toinitiate the synthesis, particularly if it has a 3′ overhang (asdemonstrated in Example 2). If the initial substrate is a linearizedplasmid backbone, the DNA synthesis method could be used to elongate theDNA molecule by one or more synthetic gene sequences and the elongatedDNA could then be (optionally amplified and) circularized into aplasmid. In general, the methods for nucleic acid synthesis describedherein enable initiation of de novo DNA synthesis from natural nucleicacid molecules; in contrast, it is not possible to directly extendnatural nucleic acid molecules by a defined sequence using thephosphoramidite method.

Strategies for Attaching a Linker to a Nucleoside Triphosphate Usefulfor Nucleic Acid Synthesis.

In some embodiments, cleavage of a linker attached to a nucleotide mayresult in the production of a natural nucleotide upon cleavage. Forexample a linker comprising a nitrobenzyl moiety alkylating an amine ofthe nucleobase may be cleaved with light, e.g. as depicted for theexocyclic amines of cytosine and adenine below, but as is applicable toany suitable nitrogen atom on any nucleobase:

In other embodiments, a linker attached to an amine of the nucleobasevia an amide linkage may be cleaved by a suitable peptidase, e.g. asdepicted for the exocyclic amines of cytosine and adenine below, but isapplicable to any suitable amino group on any nucleobase:

In such embodiments, the linker may comprise additional atoms (includedin R′ above) adjacent to the amide that increase the activity of thepeptidase towards the amide bond. In some embodiments, R′ is a peptideor polypeptide.

In some embodiments, cleavage of the linker in the deprotection step mayleave a scar that persists throughout the stepwise synthesis but that isremoved or further reduced once the stepwise synthesis is completed.This attachment strategy may enable the introduction of additionaldistance between the cleavable moiety of the linker and the nucleotide,which may be useful with certain (e.g. bulky) cleavable groups. Scarsmay be useful to prevent base pairing of incorporated nucleotides andtherefore prevent the formation of secondary structures duringsynthesis, e.g. by preventing the exocyclic amino group from engaging inbase-pairing as discussed below. Once synthesis of such a molecule iscomplete, a single “scar-removal” step can be used to preventinterference of the scars with downstream applications and to restorethe nucleic acid's base-pairing ability.

For example, acyl scars left on the exocyclic amino group of adenine,cytosine, and guanine after cleavage may hinder the formation of sometypes of secondary structures during the synthesis. After the synthesisis completed, such scars may be quantitatively removed using a mildammonia treatment (Schulhof et al., Nucleic Acids Research. 1987;15(2):397-416.) as depicted below:

An example of a linker employing these groups would be to attach alinker comprising a 2-((4-(disulfaneyl)butanoyl)oxy)acetyl group to anexocyclic amino group of a nucleobase (forming an amide), as depictedfor cytosine and adenine below:

Cleavage of the disulfide (e.g. by DTT) may result in elimination of a2-oxothiolane by intramolecular thiolactonization, leaving a glycolylscar. Another example of this strategy would be to attach a linkercomprising a photocleavable group (e.g. NPPOC) to a glycolyl of anexocyclic amino group of a nucleobase, as depicted for cytosine andadenine below:

After photo-cleavage of the linker, the bases still comprise a glycolyl(acyl) scar which may not terminate further elongation by othernucleotide-polymerase conjugates, but may ultimately be removed bytreatment with ammonia, as depicted below:

Other strategies for the attachment of the nucleoside triphosphate tothe polymerase following the above described principles may be used andwill be apparent to those skilled in the art.

Template independent polymerases may have a high tolerance for basemodifications (e.g., for TdT see Figeys et al. (Anal Chem. 1994 Dec. 1;66(23):4382-3.) and Li et al. (Cytometry. 1995 Jun. 1; 20(2):172-80.))so that certain scars may be well tolerated in following nucleic acidextension steps.

In some embodiments, linkers with multiple cleavable groups inserted intandem may be used to increase the cleavage rate compared to a linkerwith single cleavable group.

Mitigating the Inhibitory Effects of 3′ Terminal Secondary Structure

In certain embodiments, modified nucleoside triphosphates with attachedchemical moieties that prevent base pairing or the formation ofundesirable secondary structure during synthesis may be used. Suchmodifications may include, but are not limited to, N3-methylation ofcytosine, N1-methylation of adenine, 06-methylation of guanine, andacetylation of the exocyclic amine of guanine. Similar modificationswere shown to significantly enhance the rate of dGTP homopolymersynthesis using TdT (Lefler and Bollum, Journal of Biological Chemistry244.3 (1969): 594-601.). After completion of the synthesis, such basemodifications may be simultaneously removed to restore base pairing fordownstream applications. For example, N-acetylation of guanine may beremoved by ammonia treatment as described above. N3-methylation ofcytosine and N1-methylation of adenine may be removed by the enzymeAlkB, and 06-methylation of guanine may be removed by the enzyme MGMT,as depicted below:

In some embodiments, the de novo nucleic acid synthesis may be paused atan intermediate step and synthesis of complementary DNA may beperformed, e.g. by hybridization of a suitable primer (e.g. randomhexamers) that may be extended by a template-dependent polymerase usingnucleoside triphosphates. After the complementary DNA synthesis, the denovo DNA synthesis may resume, and the double stranded part of thenucleic acid may be hindered from forming secondary structures. In somecases, the complementary DNA synthesis step may comprise leaving a 3′overhang on the de novo synthesized nucleic acid to enable efficientsubsequent extensions by polymerase-nucleotide conjugates.

In some embodiments, single-stranded binding proteins (e.g. E. coli SSB)may be included in extension reactions to hinder the formation ofsecondary structures in the nucleic acid being synthesized.

Incorporation of Unnatural or Modified dNTPs.

Conjugates useful for de novo nucleic acid synthesis may comprisenucleoside triphosphate analogs, including nucleotides withoutbase-pairing ability (e.g. abasic nucleotide analogs) or nucleosidetriphosphates with base-pairing ability different from the naturalnucleotides, e.g. deoxyinosine or nitroindole nucleoside triphosphates.

Automation of De Novo Nucleic Acid Synthesis Using Polymerase-NucleotideConjugates

In some embodiments, during synthesis the nucleic acid molecules areimmobilized on a solid support that can be washed and exposed to thereaction cycle enzymes and buffers via automated liquid handingequipment. Examples of a solid support include, but are not limited to,a microtiter plate, into which reagents could be dispensed and removedby a liquid-handling robot, magnetic beads, which can be magneticallyseparated from a suspension and then resuspended in a new reagent inmicrotiter format, or an interior surface of a microfluidic device thatcan dispense the reaction cycle reagents to that location in anautomated fashion.

An application of an automated system for nucleic acid synthesisemploying conjugates comprising a polymerase and a nucleosidetriphosphate is the synthesis of 10-100 nt oligonucleotides formolecular biology applications such as PCR. Another application is thepicomole-scale or femtomole-scale synthesis of 50-500 nt or longeroligonucleotides using inkjet-based liquid handling techniques toproduce DNA molecules that serve, e.g. as input to conventional DNAassembly methods (Kosuri and Church, Nature methods 11.5 (2014):499-507.).

Single-Molecule Nucleic Acid Synthesis Using FluorescentPolymerase-Nucleotide Conjugates

In some embodiments, the DNA synthesis method can be implemented insingle-molecule format. In this approach, the reaction chamber of anautomated microfluidic device is loaded with a single primer molecule ofDNA that is iteratively extended into the desired sequence using amodified version of the reaction cycle described above (FIG. 5A). Inthis system, the conjugates comprising a polymerase and a nucleosidetriphosphate are labeled with one or more reporter molecules (e.g.fluorophores) such that once a labeled conjugate molecule has extendedthe primer by its tethered nucleotide and thereby becomes attached tothe solid support via the primer (FIG. 5B), the growing DNA moleculebecomes fluorescent. After washing away the free conjugate molecules,the polymerase attached to the primer can be detected, e.g., using afluorescence microscopy technique such as total internal reflectionfluorescence (TIRF) microscopy (FIG. 5C). After each attemptedextension, the reaction chamber is washed and imaged, and if theextension is determined to have failed, it is reattempted with the sametype of conjugate. After a successful extension is confirmed, thedeprotection reagent is introduced to the reaction chamber, cleaving thetethered labeled polymerase and thereby both deprotecting the 3′ end ofthe growing DNA molecule for subsequent extension and simultaneouslyrendering it non-fluorescent (FIG. 5D). If the deprotection fails, thefluorescence signal will remain, and the deprotection step isreattempted (FIG. 5E). These extension and deprotection checks preventthe introduction of deletion errors that inevitably accumulate duringbulk reactions that fail to go to 100% completion. An automatedsynthesizer executing this scheme will eventually synthesize one DNAmolecule with the desired sequence that can subsequently be amplified.An example of such a synthesizer is depicted in FIG. 6 . The synthesizercomprises a PDMS device with input ports for reagents, including a portfor each polymerase-nucleotide conjugate, a port for the wash buffer, aport for the deprotection buffer, and a port for the in situamplification buffer. The input ports are connected via microchannels(and, optionally, computer-actuated microvalves) to a reaction chamberwhere the synthesis takes place. The device also comprises a waste portand an output port (for collecting the synthesized products) connectedto the reaction chamber my microchannels. The device may be mounted on amicroscope suitable for single-molecule imaging, e.g. theobjective-style TIRF microscope indicated in FIG. 6 . The fluorophoresattached to the conjugates may be excited by a laser of a suitablewavelength, e.g. 532 nm; emitted light may be collected by an objectiveand imaged on a suitable detector, e.g. an electron-multiplying chargecoupled-device (EMCCD) camera connected to a computer. The computer mayexecute the synthesis scheme described above by (a) interpreting thesignals from the detector using an algorithm and (b) dispensing theappropriate reagent to the reaction chamber by actuating microvalves orpumps within or outside of the microfluidic device.

Methods for DNA Sequencing Using Polymerase-Nucleotide Conjugates.

Provided herein is a method for nucleic acid sequencing using conjugatesof a template-dependent polymerase and a nucleoside triphosphate. Themethod is analogous to Sequencing By Synthesis (SBS).

In some embodiments, the method employs an “ACGT extension reagent”comprising four conjugates with base-pairing ability equivalent to A, C,G, and T, wherein the conjugates are labeled with distinguishablelabels, e.g. distinct fluorophores. In other embodiments, the methodemploys “four distinct extension reagents” in separate containers, eachcomprising a conjugate with base-pairing ability equivalent to A, C, G,and T respectively. In some embodiments, these four extension reagentsmay be labeled for detection, e.g. with fluorophores.

In some embodiments, the method comprises: (a) immobilizing a duplexcomprising a primer and template nucleic acid on a support (b) exposingthe duplex to the “ACGT extension reagent” to extend the primer by anucleotide complementary to the template; (c) detecting the label of theattached conjugate to infer the complementary base of the template; (d)exposing the duplex to the deprotection reagent, which cleaves thelinkage between the polymerase and the added nucleotide, rendering theduplex unlabeled; and (e) repeating steps (b-d) 10 or more times todetermine the sequence of at least part of the template molecule. Anexample of this method employing polymerase-conjugates with fourdistinguishable fluorophores is depicted in FIGS. 7A-7E.

In other embodiments, the method comprises: (a) immobilizing a duplexcomprising a primer and template nucleic acid on a support (b) exposingthe duplex to the first extension reagent to extend the primer by anucleotide complementary to the template if the nucleotide of theconjugate is complementary; (c) detecting the label of the attachedconjugate to infer if an extension has occurred; (d) exposing thenucleic acid to the deprotection reagent, which cleaves the linkagebetween the polymerase and the added nucleotide, rendering the duplexunlabeled; (e) repeating steps (b-d) three more times with the remainingthree extension reagents, and (f) repeating steps (b-e) 10 or more timesto determine the sequence of at least part of the template molecule.

In some embodiments, the detectible label may be a fluorescent proteinfused to the polymerase. In other embodiments, the detectible label maybe a quantum dot that is specifically attached to the polymerase.

Particularly in embodiments that employ four distinct extensionreagents, the conjugates may be non-fluorescent or without a detectiblelabel. In such embodiments, extension may be detected by other signalsof the extension reaction such as release of H⁺ or pyrophosphate. Inother embodiments, the polymerase may be fused to a reporter enzyme suchas luciferase or peroxidase that may be detected when they produce lightby catalyzing a reaction. In other embodiments, the polymerase may befused to a nanoparticle detectible by scatting light. In otherembodiments, an otherwise unlabeled polymerase of a conjugate that hasextended a nucleic acid may be detected by a change in surface plasmonresonance.

Particularly in embodiments that employ conjugates with detectiblelabels, the method may be useful for determining the sequence ofindividual molecules, i.e. “single-molecule sequencing”.

In some embodiments, the method may additionally comprise an initialstep of making 10, 100, 1000, or more copies of the template moleculeand then applying a method described above to all copies simultaneously.

In any embodiment of the nucleic acid sequencing method, the length ofthe linker between the nucleoside triphosphate and the polymerase of theconjugates may be selected to maximize the fidelity of nucleotideincorporation by the conjugates, i.e. to minimize incorporation ofmismatched bases with the template. Likewise, in any embodiment, theconcentration of divalent cation(s) in the extension reaction (e.g.Mg²⁺) may be adjusted to maximize the fidelity of nucleotideincorporation by the conjugates.

In some embodiments, the polymerase of the conjugates may be apolymerase with “random binding order”, i.e. the primer-template duplexcan bind to the catalytic site before or after nucleoside triphosphate.

In other embodiments, the polymerase of the conjugates may be apolymerase with defined binding order, i.e. the primer-template duplexmust bind to the catalytic site before the nucleoside triphosphate. Insuch embodiments, the length of the linker between the nucleosidetriphosphate and the polymerase of the conjugates may be selected tominimize inhibition of primer-template duplex binding to the conjugate,i.e. by using a linker longer than 10 Å, or 100 Å, or 200 Å.

In any embodiment, the conjugate may comprise a reversible terminatornucleoside triphosphate.

EMBODIMENTS

Embodiment 1. A conjugate comprising a polymerase and a nucleosidetriphosphate, wherein the polymerase and the nucleoside triphosphate arecovalently linked via a linker that comprises a cleavable linkage.

Embodiment 2. The conjugate of embodiment 1, wherein the polymerase iscapable of catalyzing the addition of the nucleotide that is linked tothe polymerase to the 3′ end of a nucleic acid.

Embodiment 3. The conjugate of any prior embodiment, wherein thepolymerase is linked to the nucleoside triphosphate via a linker thathas a length in the range of 4-100 Å, and wherein the length of thelinker is sufficient for the nucleoside triphosphate to access theactive site of the polymerase.

Embodiment 4. The conjugate of any prior embodiment, wherein thenucleoside triphosphate is linked to a cysteine residue in thepolymerase.

Embodiment 5. The conjugate of any prior embodiment, wherein thecleavable linkage is a light or enzyme-cleavable linkage.

Embodiment 6. The conjugate of any prior embodiment, wherein thepolymerase is a DNA polymerase.

Embodiment 7. The conjugate of any of embodiments 1-5, wherein thepolymerase is an RNA polymerase.

Embodiment 8. The conjugate of any prior embodiment, wherein thepolymerase is a template-independent polymerase.

Embodiment 9. The conjugate of any of embodiments 1-7, wherein thepolymerase is a template-dependent polymerase.

Embodiment 10. The conjugate of any prior embodiment, wherein thenucleoside triphosphate or the polymerase comprises a fluorescent label.

Embodiment 11. The conjugate of any prior embodiment, wherein thenucleoside triphosphate is a deoxyribonucleoside triphosphate.

Embodiment 12. The conjugate of any prior embodiment, wherein thenucleoside triphosphate is a ribonucleoside triphosphate.

Embodiment 13. A set of conjugates of any prior embodiment, wherein theconjugates correspond to G, A, T and C and are in separate containers.

Embodiment 14. A method of nucleic acid synthesis, comprising:

incubating a nucleic acid with a first conjugate, wherein the firstconjugate is a conjugate of any prior embodiment and the incubating isdone under conditions in which the polymerase catalyzes the covalentaddition of the nucleotide of the first conjugate onto the 3′ hydroxylof the nucleic acid, to make an extension product.

Embodiment 15. The method of embodiment 14, wherein nucleic acid istethered to a support.

Embodiment 16. The method of embodiment 14 or 15, wherein the methodcomprises, after addition of the nucleotide onto the nucleic acid,cleaving the cleavable linkage of the linker, thereby releasing thepolymerase from the extension product.

Embodiment 17. The method of embodiment 16, wherein the cleavablelinkage is an enzyme- or light-cleavable linkage and the cleavingcomprises exposing the extension product to an enzyme or to light.

Embodiment 18. The method of embodiments 16 or 17, wherein the cleavageof the cleavable linkage deprotects the added nucleotide to produce adeprotected extension product.

Embodiment 19. The method of embodiment 18, further comprising, afterdeprotection of the added nucleotide:

incubating the deprotected extension product with a second conjugate,wherein the second conjugate is a conjugate of any of embodiments 1-12and the incubating is done under conditions in which the polymerasecatalyzes the covalent addition of the nucleotide of the secondconjugate onto the 3′ end of the deprotected extension product.

Embodiment 20. The method of any of embodiments 14-19, wherein themethod comprises:

(a) incubating a nucleic acid with a first conjugate of any ofembodiments 1-12 under conditions in which the polymerase catalyzes thecovalent addition of the nucleotide of the first conjugate onto the 3′hydroxyl of the nucleic acid, to make an extension product;

(b) cleaving the cleavable linkage of the linker, thereby releasing thepolymerase from the extension product and deprotecting the extensionproduct;

(c) incubating the deprotected extension product with a second conjugateof any of embodiments 1-12 under conditions in which the polymerasecatalyzes the covalent addition of the nucleotide of the secondconjugate onto the 3′ end of the extension product, to make a secondextension product;

(d) repeating steps (b)-(c) on the second extension product multipletimes to produce an extended nucleic acid of a defined sequence.

Embodiment 21. The method of embodiment 20, wherein the nucleotide is areversible terminator, and wherein deprotection of the extension productcomprises removal of the blocking group of the reversible terminator.

Embodiment 22. The method of any of embodiments 14-21, wherein thenucleic acid is an oligonucleotide.

Embodiment 23. A method of sequencing, comprising:

incubating a duplex comprising a primer and a template with acomposition comprising a set of conjugates of embodiment 13, wherein theconjugates correspond to G, A, T (or U) and C and are distinguishablylabeled;

detecting which nucleotide has been added to the primer by detecting alabel that is tethered to the polymerase that has added the nucleotideto the primer;

deprotecting the extension product by cleaving the linker; and

repeating the incubation, detection and deprotection steps to obtain thesequence of at least part of the template.

Embodiment 24. The method of embodiment 23, wherein the method ofsequencing is a method of DNA sequencing.

Embodiment 25. The method of embodiment 23, wherein the method ofsequencing is a method of RNA sequencing.

Embodiment 26. The method of any of embodiments 23-25, wherein thenucleotide is a reversible terminator, and wherein deprotection of theextension product comprises removal of the blocking group.

Embodiment 27. A reagent set, comprising:

a polymerase that has been modified to contain a single cysteine on itssurface; and

a set of nucleoside triphosphates, wherein each of the nucleosidetriphosphates is linked to a sulfhydryl-reactive group.

Embodiment 28. The reagent set of embodiment 27, wherein the nucleosidetriphosphates correspond to G, A, T (or U) and C.

Embodiment 29. The reagent set of any of embodiments 27-28, wherein thenucleoside triphosphates are reversible terminators.

Embodiment 30. The reagent set of any of embodiments 27-29, wherein thenucleoside triphosphates comprise a linker that has a length in therange of 4-100 Å.

EXAMPLES

Aspects of the present teachings can be further understood in light ofthe following examples, which should not be construed as limiting thescope of the present teachings in any way.

Example 1 Tethered Nucleotide Incorporation by Polymerase-NucleotideConjugates Employing a Linker Cleavable by Reducing Agents

1. Generation of Polymerase (TdT) Mutants with Various AttachmentPositions for the Linker.

A gBlock coding for the Mus musculus TdT amino acid sequence used byBoule et al. (Molecular biotechnology 10.3 (1998): 199-208.) was orderedfrom IDT (Coralville, Iowa). The sequence was cloned into a pET19bvector, fusing the N-terminal his-tag of the vector to the protein usingisothermal assembly. QuickChange PCR was used to generate a TdT mutantlacking all surface cysteines that might allow incorporation of tethereddNTPs (TdT5cysX). The cysteines in positions 188, 302, and 378 weremutated into alanine, the cysteines in positions 216 and 438 weremutated into serine (positions refer to the numbering in PDB structure4127). TdT mutants containing one surface cysteine close to thecatalytic site were generated by QuickChange PCR on TdT5cysX. Cysteineswere inserted in positions 188, 302, 180 or 253, respectively.

Listed below is the amino acid sequence of the “wildtype” TdT proteinused in this example prior to the mutation of cysteine residues (TdTwt):

(SEQ ID NO: 1) MGHHHHHHHHHHSSGHIDDDDKHMSQYACQRRTTLNNHNQIFTDAFDILAENDEFRENEGPSLTFMRAASVLKSLPFTIISMKDIEGIPNLGDRVKSIIEEIIEDGESSAVKAVLNDERYKSFKLFTSVFGVGLKTSEKWFRMGFRTLSNIRSDKSLTFTRMQRAGFLYYEDLVSRVTRAEAEAVGVLVKEAVWASLPDAFVTMTGGFRRGKKTGHDVDFLITSPGATEEEEQQLLHKVISLWEHKGLLLYYDLVESTFEKLKLPSRKVDALDHFQKCFLILKLHHQRVDSDQSSWQEGKTWKAIRVDLVVCPYERRAFALLGWTGSRQFERDLRRYATHERKMIIDNHALYDKTKRIFLEAESEEEIFAHLGLDYIEPWERNA.

As described above, plasmids coding for 6 TdT mutants with varyingcysteine residues (therefore with different attachment positions for alinker) were generated:

(a) A plasmid coding for “wildtype” TdT with 7 cysteines (TdTwt)

(b) A plasmid coding for a TdT mutant with no surface cysteines and only2 cysteines that are both buried (TdT5cysX)

(c) Four plasmids coding for TdT mutants with 2 buried cysteines plusone exposed surface cysteine in different positions (188, 180, 253, and302) referred to herein as TdTcys188, TdTcys 180, TdTcys253, andTdTcys302

2. Protein Expression and Purification of the Mutants.

TdT expression was performed using Rosetta-gami B(DE3)pLysS cells(Novagen) in LB media containing antibiotics for all four resistancemarkers of the cells (Kan, Cmp, Tet, and Carb, which is introduced bythe pET19 vector). An overnight culture of 50 mL was used to inoculate a400 mL expression culture with 1/20 vol. Cells were grown at 37° C. and200 rpm shaking until they reached OD 0.6. IPTG was added to a finalconcentration of 0.5 mM and the expression was performed for 12h at 30°C. Cells were harvested by centrifugation at 8000 G for 10 min andresuspended in 20 mL buffer A (20 mM Tris-HCl, 0.5 M NaCl, pH 8.3)+5 minimidazole. Cell lysis was performed using sonication followed bycentrifugation at 15000 G for 20 min. The supernatant was applied to agravity column containing 1 mL of Ni-NTA agarose (Qiagen). The columnwas washed with 20 volumes of buffer A+40 mM imidazole, and boundprotein was eluted using 4 mL buffer A+500 mM imidazole. The protein wasconcentrated to ˜0.15 mL with Vivaspin 20 columns (MWCO 10 kDa,Sartorius) and then dialyzed against 200 mL TdT storage buffer (100 mMNaCl, 200 mM K2HPO4, pH 7.5) over night using Pur-A-Lyzer™ Dialysis KitMini 12000 tubes (Sigma).

All 6 TdT mutants with varying cysteine residues were expressed andpurified.

3. Attachment of Tethered Nucleoside Triphosphates to the Polymerase

To prepare TdT-dUTP conjugates, the linker-nucleotide OPSS-PEG4-aa-dUTPwas first synthesized and then reacted with TdT. OPSS-PEG4-aa-dUTP wassynthesized by reacting amino-allyl dUTP (aa-dUTP) with theheterobifunctional crosslinker PEG4-SPDP (FIG. 3A). The reactioncontained 12.5 mM aa-dUTP, 3 mM PEG4-SPDP crosslinker and 125 mM sodiumbicarbonate (ph 8.3) in a volume of 8 μL and was performed at RT for 1h.The reaction was quenched by the addition of 1 μL of 100 mM glycine inPBS for 10 min. The buffer was adjusted to the OPSS-labeling conditionsby the addition of 1 μL 10× TdT storage buffer and then 70-100 μgpurified protein in 40 μL 1× TdT storage buffer were added. The reactionto attach the linker-nucleotide to TdT was performed at RT for 13h.Removal of free (i.e. unattached) linker-nucleotides was conducted usingthe Capturem His-Tagged Purification Miniprep Kit (Clonetech).Purification resulted in protein concentrations between 0.2 and 0.4μg/μL. Dialysis against 100 mL 1× TdT reaction buffer (NEB) wasperformed in Pur-A-Lyzer™ Dialysis Kit Mini 12000 tubes for 4h.

The scheme for the preparation of the polymerase-nucleotide conjugatesis shown in FIG. 3 . First, the aminoallyl dUTP (aa-dUTP) is reactedwith the heterobifunctional amine-to-thiol crosslinker PEG4-SPDP (panelA) to form the thiol-reactive linker-nucleotide OPSS-PEG4-aa-dUTP (panelB). OPSS-PEG4-aa-dUTP can then be used to site-specifically label TdT atsurface cysteine residues (panel C) via disulfide bond formation.

All 6 TdT mutants with varying cysteine residues were separately exposedto OPSS-PEG4-aa-dUTP tethering reactions.

(a) TdTwt contains five surface cysteine residues that result inlabeling with up to five OPSS-PEG4-aa-dUTP moieties.

(b) TdT5cysX only contains two buried but no surface cysteine residues,presumably not resulting in substantial labeling by OPSS-PEG4-aa-dUTP.

(c) TdTcys188, TdTcys180, TdTcys253, and TdTcys302 have a single surfacecysteine that can be labeled with a single OPSS-PEG4-aa-dUTP moiety (atresidue position 188, 180, 253, and 302, respectively). These TdTmutants also contain the two buried cysteines present in TdT5cysX thatpresumably are not labeled.

4. Generation of the Ladder of Elongation Product Standards.

The ladder of linked incorporation product standards was generated byincorporating free OPSS-PEG4-aa-dUTP using TdT. The reaction tosynthesize OPSS-PEG4-aa-dUTP was performed by mixing 6 μL 50 mM aa-dUTP,5 μL 180 mM PEG4-SPDP, 5 μL 1 M NaHCO₃ and 4 μL ddH₂O. The reaction wasincubated at RT for 1h and another 5 μL of 180 mM PEG4-SPDP was added.After 1h, the reaction was quenched using 5 μL of 100 mM glycine in PBS.To achieve varying numbers of incorporations, 6 TdT incorporationreactions using free OPSS-PEG4-aa-dUTP as substrate were performed. Thereactions contained 1.5 μL 10×NEB TdT reaction buffer, 1.5 μL NEB TdTCoCl₂, 1.5 μL 10 μM 5′-FAM labeled 35-mer dT-oligonucleotide(5′-FAM-dT(35)), 1 μL 10 mM OPSS-PEG4-aa-dUTP, 4.5 μL ddH₂O and variedin their TdT concentration (100, 50, 25, 12.5, 6.3, 3.13 units of NEBTdT in 5 μL 1×NEB reaction buffer). Reactions were performed for 5 mM at37° C. and stopped by the addition of 0.3 mM EDTA. Before running theladder on the polyacrylamide gel, reaction products were mixed with anequal volume of 2× Novex Tris-Glycine buffer+1% v/v f3-mercaptoethanoland heated to 95° C. for 5 mM.

The 5′-FAM-dT₍₆₀₎ oligo was extended by 0 to 5 or more OPSS-PEG4-aa-dUMPnucleotides. After reduction of the ladder in the loading dye, theHS-PEG4-aa-dUTP elongation product standards (structure of oneHS-PEG4-aa-dUTP elongation product is depicted in FIG. 3E) were resolvedon a polyacrylamide gel (FIGS. 8A and B, lanes labeled “L”). The ladderwas used to identify the cleaved products of primer elongation reactionswith polymerase-nucleotide conjugates by comparison of the migration ofthe elongation product bands to the ladder bands.

5. Incorporation of Tethered Nucleoside Triphosphates into a NucleicAcid.

The OPSS-PEG4-aa-dUTP conjugates of TdTwt, TdT5cysX, TdTcys188,TdTcys180, TdTcys253, and TdTcys302 were reacted with 5′-FAM-dT(35).Reactions that are shown in FIG. 8A contained 1 μL of 5 μM5′-FAM-dT(35), 17 μL of the purified, conjugated TdT variants in 1× TdTreaction buffer (NEB) and 2 μL of 2.5 mM CoCl₂. The reactions wereperformed for 20 sec at 37° C. and then quenched by the addition of 33mM EDTA. Reactions shown in FIG. 8B contained 1.5 μL of 5 μM5′-FAM-dT(35), 1 μL 10×NEB reaction buffer, 1.5 μL of 2.5 mM CoCl₂, 5 μLof the respective TdT conjugate in 1× TdT buffer and 6 μL ddH₂O.Reactions were performed at 37° C. for 40 sec, quenching was performedby the addition of 33 mM EDTA. To prepare the reactions for the gel,samples were mixed with the equivalent volume of 2× Novex Tris-GlycineSDS Sample Buffer (Thermo Scientific), or with 2× Novex Tris-Glycinebuffer+1% v/v 2-mercaptoethanol (BME), respectively. All samples wereheated to 95° C. for 5 min and run on SDS.

As shown in chemical detail in FIG. 3 , TdT conjugates ofOPSS-PEG4-aa-dUTP (panel C) can incorporate a tethered nucleotide into aprimer, which results in covalent attachment of the TdT moiety to theelongated primer (panel D). For detection purposes the primer can belabeled at its 5′ end with a fluorescent dye such as6-carboxyfluorescein (FAM). The primer-polymerase complexes can bedissociated by exposure to (WE, which cleaves the disulfide bond betweenthe incorporated nucleotide and TdT, releasing free TdT and a primerthat is elongated by a dUMP harboring a HS-PEG4-aa scar (panel E).

To demonstrate that polymerase-nucleotide conjugates add their tetherednucleotides to an oligonucleotide, TdT conjugates of OPSS-PEG4-aa-dUTPwere incubated with a 5′ FAM-labeled dT(35) primer. The reaction wasperformed with conjugates of TdTwt, which have multiple tetherednucleotides, conjugates of TdT5cysX, which do not have tetherednucleotides, and conjugates of TdTcys302 which has a single nucleotidetethered to the cysteine at position 302. As described above, thereactions were stopped and the products were resolved by SDS-PAGE (FIG.8A). TdTwt and TdTcys302 conjugates added their tethered nucleotide(s)to the 3′ end of the primer and became covalently linked forming apolymerase-primer complex, as indicated by much slower migration of thebands on SDS-PAGE (lanes 3 and 11, respectively) compared to the primer(lanes labeled “P”: 2, 6, and 10). In contrast, no shift in migrationwas seen with TdT5cysX (lane 7) that does not comprise tetherednucleotides. Upon addition of the disulfide-cleaving reagent2-mercaptoethanol, the primer-TdT complexes dissociated, as indicated byrestored migration of the bands (lanes labeled “B”: 4, 8, and 12 forTdTwt, TdT5cysX, and TdTcys302 respectively). The extension of theprimer can be referenced to the ladder of product standards (laneslabeled “L”: 1, 5, 9, and 13). Conjugates of TdTwt incorporated multipletethered nucleotides, yielding a primer that was extended by up to 5scarred dUMP nucleotides (lane 4). Conjugates of TdTcys302 predominantlyextended the primer by a single scarred dUMP nucleotide (lane 12), andconjugates of TdT5cysX did not extend the primer (lane 8).

These data show that the polymerase moiety of polymerase-nucleotideconjugates can incorporate one or more tethered nucleoside triphosphatesinto a primer. They also show that conjugates labeled with a singlenucleotide triphosphate can perform a single elongation of a primer andthat further elongations of the primer by other polymerase-nucleotideconjugates can be hindered, enabling elongation of a nucleic acid by asingle nucleotide.

To demonstrate that functional polymerase-nucleotide conjugates can begenerated using a variety of attachment positions on the polymerase,OPSS-PEG4-aa-dUTP conjugates of TdT mutants with a single surfacecysteine at positions 188, 302, 180, and 253 (TdTcys188, TdTcys302,TdTcys180, and TdTcys253, respectively) were used to extend a primer bya single nucleotide. The conjugates were separately exposed to a 5′fluorescently-labeled poly-dT primer. Separation of the products onSDS-PAGE (FIG. 8B) revealed that all four conjugates were able toincorporate a tethered nucleotide to the 3′ end of the primer, asindicated by the formation of a much slower migrating band correspondingto the polymerase-primer complex (lanes 4-7, respectively) compared toprimer band (fastest migrating band of lanes labeled “L”: 1, 8, and 15).Upon cleavage of the linker by 2-mercaptoethanol (BME), thepolymerase-primer complex dissociated, leaving a primer that had beenpredominantly extended by 1 scarred dUMP nucleotide (lanes 11-14,respectively) as identified by comparison with the ladder of productstandards (lanes labeled “L”).

These data show that the principle of tethering a single nucleotide to apolymerase for achieving single nucleotide elongation of a nucleic acidis generalizable across attachment points on the polymerase.

Example 2 Synthesis of a Defined DNA Sequence UsingPolymerase-Nucleotide Conjugates Employing a Light-Cleavable Linker

-   -   1. Generation of an MBP-TdT Fusion Protein with Only One Surface        Exposed Cysteine (TdTcys).

The sequence encoding Maltose Binding Protein (MBP) was amplified frompMAL-c5X (NEB) and N-terminally fused to the TdTcys302 construct used inExample 1 using isothermal assembly. The resulting MBP-TdT fusionprotein (herein referred to as TdTcys) was used throughout Example 2.

Protein Sequence of TdTcys:

(SEQ ID NO: 2) MGHHHHHHHHHHSSGHIDDDDKHMMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMSMGGRDIVDGSEFSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSALAFMRASSVLKSLPFPITSMKDTEGIPSLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYADILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMSPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA.

-   -   2. Protein Expression and Purification Based on Nickel Affinity        Chromatography Followed by Anion Exchange Chromatography.

Unless mentioned otherwise, E. coli BL21(DE3) harboring pET19-TdTcys wasgrown in LB (Miller) with 100 μg/mL carbenicillin while shaking at 200RPM. An overnight culture was diluted 1/60 into 400 mL LB in a 2 L flaskwithout baffles and grown at 37° C. until an OD₆₀₀ of 0.40-0.45 wasreached. The flasks were then cooled down to RT for 45 min withoutshaking and then shaken at 15° C. for 45 min. Protein expression wasinduced with IPTG (final conc. 1 mM) and cells were grown overnight at15° C. and harvested by centrifugation. All protein purification stepswere performed at 4° C. Cells were lysed in Buffer A (20 mM Tris-HCl, pH8.3, 0.5 M NaCl)+5 mM imidazole and the lysate was subjected to nickelaffinity chromatography (HisTrap FF 5 mL, GE Healthcare) with animidazole gradient (Buffer A+5 mM imidazole to Buffer A+500 mMimidazole). Fractions with sufficient purity were pooled, diluted 1:40into 20 mM Tris-HCl, pH 8.3 and subjected to anion-exchangechromatography (HiTrap Q HP 5 mL, GE Healthcare) in 20 mM Tris-HCl usinga gradient of 0 to 1 M NaCl. The protein eluted at 200 mM NaCl. Theprotein was stored at −20° C. after the addition of 50% glycerol.

3. Preparation of TdT-dNTP Conjugates.

Propargylamino-dNTPs (pa-dNTPs) were coupled to the photocleavable NHScarbonate-maleimide crosslinker BP-23354 (FIG. 4A) in a 35 μL reactioncontaining 3.3 mM of the respective pa-dNTP, 6.6 mM linker, 66 mM KH₂PO₄at pH 7.5 and 33 mM NaCl, for 1 h at RT with gentle vortexing. Thereaction was split up into 7.5 μL aliquots, triturated with ethylacetate (˜2 mL) and centrifuged at 15,000 g to pellet thelinker-nucleotides. The supernatant was removed and the pellets weredried in a speedvac at RT for 8 min. The pellets were resuspended in 2.5μL of water and the linker-nucleotides were added to 20 μL of TdTcysprotein prepared as described above plus 2.5 μL of pH 6.5 buffer (2 MKH₂PO₄, 1 M NaCl) and incubated for 1 h at RT. TdTcys-linker-dNTPconjugates (also referred to as TdT-dNTP conjugates) were then purifiedusing amylose resin (NEB) in 0.8 mL spin columns (Pierce). All reagentsand buffers were precooled on ice. The 25 μL TdTcys linker-nucleotideconjugation reaction was diluted into 400 μL Buffer B (200 mM KH₂PO₄ pH7.5, 100 mM NaCl) and split across two purification columns, eachcontaining 250 μL amylose resin in Buffer B. After 10 min of proteinbinding with gentle vortexing, the column was washed twice with BufferB, and then twice with 1×NEB TdT reaction buffer (50 mM potassiumacetate, 20 mM Tris-acetate, 10 mM magnesium acetate, pH 7.9). Washingwas performed by adding 500 μL buffer to the column, incubating for 1min on a shaker block to mix resin and buffer (800 RPM), followed bycentrifugation at 50 g for 1 min. Elution was performed by twiceresuspending the resin in 150 μL TdT reaction buffer+10 mM maltose withshaking for 5 minutes followed by centrifugation. The eluates werecombined and concentrated in 30 kDa MWCO columns, diluted 1:10 with TdTreaction buffer, and then concentrated to ˜2.5 μg/μL.

Analogs of the four nucleotides dATP, dCTP, dGTP, and dTTP wereseparately coupled to the photocleavable crosslinker and tethered toTdT. The different polymerase-nucleotide conjugates were purified usingamylose affinity chromatography.

4. Capillary Electrophoresis Analysis and Ladder Generation.

Capillary electrophoresis (CE) analysis throughout Example 2 was run onan ABI 3730x1 DNA Analyzer. GeneScan Liz600 v1.0 (Thermo) was added toall samples for use as internal size standards. Ladders (size standards)for 5′-FAM labeled 60-mer dT-oligonucleotide (5′-FAM-dT(60)) extensionproducts were generated by the incorporation of free pa-dNTPs with TdT.Reactions contained 100 nM 5′-FAM-dT(60), 100 μM of one type of pa-dNTP,1×RBC and either 0.05 U/μL or 0.03 U/μL NEB TdT. Reactions wereperformed at 37° C. Aliquots were taken after 2, 5 and 10 min andquenched with EDTA to a final concentration of 33.3 mM. Quenched sampleswere then acetylated using NHS-acetate, purified Oligo Clean &Concentrator kit (“OCC”, Zymo Research) and analyzed by capillaryelectrophoresis. Samples with detectable peaks for 5′-FAM-dT(60) as wellas the +1 and +2 pa-dNTP extension products were chosen as sizestandards (ladders).

5. Demonstration of Two Reaction Cycles on PAGE and CapillaryElectrophoresis.

Throughout the experiment, primer extension reactions were performed for2 min at 37° C. and quenched by the addition of an equal volume of 200mM EDTA. All reactions contained 50 nM 5′-FAM-dT(60),TdTcys(-linker)/TdT-dCTP at 0.25 mg/mL and 1×RBC (1×NEB TdT reactionbuffer, 0.25 mM cobalt). Light induced cleavage of the linker wasperformed using a Benchtop 2UV Transilluminator (UVP, LLC) on the 365 nmsetting for 1 h on ice. Measured irradiance was approximately 5 mW/cm².Two cycle experiment: a reaction containing TdT-dCTP conjugate and5′-FAM-dT(60) was performed and the product was cleaved with 365 nmlight. The oligo was then purified (Zymo OCC), and subjected to anotherreaction with TdT-dCTP, again followed by a light cleavage step.Aliquots were taken after both extension reactions (for PAGE) and afterboth light cleavage reactions (for PAGE and CE). For the controlexperiment (“control unlinked”), TdT-dCTP conjugate was cleaved byirradiation with 365 nm light for 1 h on ice to generate an equimolarmix of unlinked TdTcys(-linker)+pa-dCTP. The products were then reactedwith 5′-FAM-dT(60). Aliquots for PAGE and CE were taken after quenchingthe reaction with EDTA. Sample preparation: all CE samples wereacetylated using 20 mM NHS-acetate in bicarbonate buffer prior toanalysis. Samples were combined with SDS loading dye and analyzed byPAGE, the gel was imaged for green fluorescence (of the 5′FAM-labeledprimer) and, after staining with Lumitein UV (Biotium), imaged for redfluorescence (total protein).

Exposure of a 5′ PAM-labeled oligonucleotide primer to TdT-dCTPconjugate resulted in a covalent complex visible on SDS-PAGE containingboth the DNA primer and the protein (FIG. 9A). Irradiation of thecomplex with 365 nm UV light cleaved the linker and thereby dissociatedthe complex, releasing a primer that had been predominantly extended bya single scarred dCMP nucleotide (FIG. 9B). This product was exposed tofresh TdT-dCTP, and it again formed a primer-TdT complex, which againwas dissociated by UV irradiation, releasing a primer that had now beenextended by two nucleotides. In contrast, no primer-TdT complexformation was observed in a control reaction in TdT-dCTP was irradiatedprior to addition of the DNA primer (FIG. 9A); instead, the controlreaction produced a variety of primer extension products (FIG. 9B)consistent with TdT-catalyzed incorporation of free nucleotides.

These data show that the process of extending a primer by one nucleotideusing a polymerase-nucleotide conjugate can be repeated to elongate aprimer by a defined sequence.

6. Rapid Single Nucleotide Incorporation by TdT-dCTP, TdT-dGTP,TdT-dTTP, and TdT-dATP.

Oligonucleotide extension yield by 1.5 mg/mL TdT-dNTP conjugate wasmeasured at 8, 15, and 120 seconds. Reactions were performed at 37° C.by adding 4.5 μL of TdT-dNTP conjugate (2 mg/mL) to 1.5 μL 5′-FAM-dT(60)(100 nM, final 25 nM), both in 1×RBC. After rapid mixing, 4.5 μL of thereaction were quenched in 18 μL QS (94% HiDi formamide, 10 mM EDTA)after 8 or 15 sec. The remaining reaction volume was quenched with 6 μLQS after 2 min. All samples were irradiated at 365 nm on a Benchtop 2UVTransilluminator (UVP, LLC) for 30 min to cleave the linker. Cleavageproducts were diluted with wash buffer (0.67 M NaH₂PO₄, 0.67 M NaCl,0.17 M EDTA, pH 8) and captured onto DynaBeads M-280 StreptAvidin(Thermo) saturated with a 5′ biotinyl dA(60) oligo, washed, acetylatedusing 100 mM NHS-acetate in bicarbonate buffer, and eluted with 75%deionized formamide for CE.

The CE data in FIG. 10 show that the primer was transformed into thesingly-extended complex in less than 20 sec of incubation with TdT-dCTP,TdT-dGTP, TdT-dTTP, and TdT-dATP. These results demonstrate thatpolymerase-nucleotide conjugates can elongate a primer by one nucleotiderapidly and with excellent yield.

7. Cyclic Synthesis of a Defined DNA Sequence.

Four iterations of nucleic acid extension and deprotection wereperformed using TdT-dNTP conjugates at 0.25 mg/mL. Extension reactionswere performed with 2 min incubations at 37° C. in 1×RBC and werequenched by the addition of an equal volume of quenching buffer (250 mMEDTA, 500 mM NaCl). Cleavage of the linker was performed by irradiationat 365 nm. The first extension reaction contained TdT-dCTP and 50 nMoligo C1 (/5Phos/UTGAAGAGCGAGAGTGAGTGA/iFluorT/CATTAAAGACGTGGGCCTGGAttt(SEQ ID NO: 3) where/5Phos/refers to a 5′ phosphorylation, /iFluorT/refers to a dT nucleotide base-modified with fluorescein). Afterphotolysis, the extension product was purified (Zymo OCC), and therecovered DNA was subjected to the next extension step with TdT-dTTP.Two more cycles were performed with TdT-dATP and subsequently withTdT-dGTP, and the ultimate product was T-tailed using TdT and freedTTP+ddTTP at a ratio of 100:1. The tailed product was thenPCR-amplified using HotStart Taq (NEB) with primers C2(GTGCCGTGAGACCTGGCTCCTGACGATATGGATaagcttTGAAGA GCGAGAGTGAGTGA; SEQ IDNO:4) and C3 (AAAAgaattcAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAA; SEQ ID NO:5) (PCR program: Initial cycle of 98° C. for 2 min, 49°C. for 20 sec, 68° C. for 5 min, then 30 cycles of three step protocol:98° C. for 30 sec, 49° C. for 20 sec, 68° C. for 30 sec). The PCRproduct was inserted into the pUC19 plasmid using EcoRI and HindIIIsites that were introduced by the PCR primers. The plasmid wastransformed into DH10B cells and the plasmids of single colonies wereextracted after overnight growth in LB and sequenced.

As described in detail above, four iterations of the reaction cycle wereperformed on a starter DNA molecule, the tailed products werePCR-amplified, and the amplicon was cloned for sequencing as depicted inFIG. 11A. Of 35 clones sequenced, 31 (89%) contained the complete5′-CTAG-3′ sequence (FIG. 11B), implying an average stepwise yield of97%. These data show that polymerase-nucleotide conjugates can be usedin a cyclic process to write a defined sequence of DNA with excellentstepwise yield.

Example 3 Incorporation of Free dNTPs into a Primer Already Tethered toa Polymerase

The experiment was performed using TdT-dCTP conjugates as prepared inExample 2. Capillary electrophoresis analysis was also performed asdescribed in Example 2, and the same oligonucleotide ladder was used assize reference. 5′-FAM-dT₆₀ (50 nM) was incubated with TdT-dCTP (0.25mg/mL) for 120 sec at 37° C. in 1×RBC to yield a primer-polymerasecomplex and the reaction was split into aliquots. One aliquot wasquenched by the addition EDTA to a final concentration of 100 mM.Another aliquot was diluted 10-fold with 1×RBC. Another aliquot wasdiluted 10-fold with 1×RBC containing free pa-dCTP to a finalconcentration of 500 μM pa-dCTP. After incubation at 37° C. for another60 sec, both reactions were quenched by the addition of EDTA to a finalconcentration of 100 mM. Light induced cleavage of the linker for allsamples was performed using a Benchtop 2UV Transilluminator (UVP, LLC)on the 365 nm setting for 1 h on ice. The samples were acetylated using20 mM NHS-acetate in bicarbonate buffer, purified and analyzed by CE.During the initial incubation of the primer with TdT-dCTP, aprimer-polymerase complex forms, as indicated by the extension of theprimer by a single nucleotide observed by CE analysis (FIG. 12 ). In thereaction allowed to proceed for another 60 sec, no further extensions ofthe primer are detected. However, in the reaction allowed to proceed foranother 60 sec with free nucleoside triphosphates (pa-dCTP) added,further extensions of the tethered primer were observed.

These results show that a nucleic acid-polymerase complex that is formedupon tethered incorporation of a nucleoside triphosphate by apolymerase-nucleotide conjugate may still be able to incorporate freenucleoside triphosphates.

Example 4 Conversion of a “Scarred” Polynucleotide into Natural DNA

Fluorescent primers were prepared by labeling amine-containing oligoswith 4.5 mM fluorophore NHS ester in sodium bicarbonate buffer followedby OCC. A 639 nt DNA product containing deoxyuridine bases was obtainedby 35 cycles of PCR using Phusion U (Thermo) following themanufacturer's instructions (two step protocol: 98° C. denaturation for10 sec, 72° C. annealing/extension for 1 min) from the plasmid templatepMal-c5x (NEB) using primers PA1 (/5Phos/cattaaagacgtgggcgtgga; SEQ IDNO:6) and PA2 (t*t*t/iUniAmM/tgtgaaatccttccctcgatcc; SEQ ID NO:7).Primer PA1 is 5′ phosphorylated; primer PA2 contains an internal aminogroup (/iUniAmM/) labeled with Cy3 NHS (GE Healtcare) and begins withtwo phosphorothioate linkages (*) to render it exonuclease-resistant.The PCR product was purified from a 1% TAE-agarose gel and ˜6.7 μg ofproduct was digested with 5 U of Lambda exonuclease (NEB) in a 100 μLreaction for 20 min at 37° C. to isolate the Cy3-labeled strand.Digestion products were purified by OCC and then hybridized at ˜1 μMwith equimolar 5′FAM-labeled primer PA3(/5AmMC6/CAACACACCACCCACCCAACcgcagatgtccgctttctgg (SEQ ID NO:14);/5AmMC6/refers to an aminohexyl modification of a 5′ phosphate) in 1×CutSmart buffer (NEB) by heating to 85° C. and cooling to 25° C. at 1°C./min. N-acetyl propargylamino dNTPs were prepared by acetylating 13 mMpropargylamino dNTPs with 25 mM NHS acetate in 100 mM sodium bicarbonatebuffer and quenched with glycine to 25 mM final. The primer was thenelongated using 7.5 U of Klenow(exo-) (NEB) in 30 μL reactions at 37° C.with 200 μM (each) N-acetyl propargylamino dNTPs (reaction ii) orwithout any dNTPs (reaction i). After 1 hour, 3 μL of 2.5 mM (each)ddNTPs (Affymetrix) were added to both reactions for an additional 15min incubation followed by inactivation of the polymerase by heating to75° C. for 20 minutes. The products were then immediately digested in 50μL reactions using 5 U of USER Enzyme (NEB) at 37° C. for 1 hour toremove the dU-containing ssDNA template. Digestion products werepurified by OCC and propargylamino dNTP-dependent elongation of the5′FAM-labeled primer and USER digestion of the Cy3-labeled template wereconfirmed by CE. Both products were then used as templates forcomplementary DNA synthesis (“reading”) by 5 U of Taq (Thermo) using 200μM (each) natural dNTPs and 200 nM of the 5′ Cy3-labeled primer PA4(/5AmMC6/CGACTCACCTCACGTCCTCAtgtgaaatccttccctcgatcc; SEQ ID NO:8) in a20 μL reaction by heating to 95° C. for 2 min and then incubating at 45°C. After 30 minutes, 1 μL of 2.5 mM (each) ddNTPs was added to bothreactions for an additional 15 min incubation, and the DNA products werepurified. Equal volumes of both reading products were then analyzed byqPCR on a CFX96 instrument (Bio-Rad), using Phusion HS II (Thermo) with1× EvaGreen (Biotium) and primers PA5 (ttttGAATTCCAACACACCACCCACCCAAC;SEQ ID NO:9) and PA6 (ttttAAGCTTCGACTCACCTCACGTCCTCA; SEQ ID NO:10) for30 cycles of 98° C. for 5 sec, 67° C. for 15 sec, and 72° C. for 30 sec.qPCR products from reaction ii were inserted into a pUC19 plasmid usingEcoRI and HindIII sites introduced by the PCR primers and 81 clones weresequenced as described above.

The dNTP analogs used in this example contain a propargylamino groupextending from the nucleobase (5 position of pyrimidines, 7 position of7-deazapurines) which is the same moiety the polymerase-nucleotideconjugates from Example 2 leave as a scar. The propargylamino moiety wasfurther derivitized by N-acetylation. To demonstrate that DNA comprisingscarred bases as produced in Example 2 can serve as template foraccurate synthesis of complementary DNA by a template-dependentpolymerase using natural dNTPs, in this example a DNA moleculecontaining 141 sequential 3-acetamidopropynyl (i.e. N-acetylatedpropargylamino) nucleotides was prepared. This DNA product was isolatedand used as template for PCR (FIGS. 13A-13C). The PCR product wasinserted into a plasmid, cloned into E. coli and 81 colonies weresequenced. 5 errors were found, implying an error rate for the synthesisof natural DNA from the 3-acetamidopropynyl modified template ofapproximately 6×10⁻⁴/nt.

This data shows that scarred nucleic acids (in this case apolynucleotide harboring a moiety derivable from the propargylamino scarfrom Example 2) can be PCR-amplified with high fidelity, therebygenerating natural DNA that can be used in biological applications.

Example 5 Synthesis of a 10-Mer

Ten extensions of the 3′ overhang of a double-stranded DNA molecule wereperformed using TdT-dNTP conjugates as prepared in Example 2. The doublestranded DNA used as initial substrate was prepared from a ˜350 bp PCRproduct derived from the pET19b plasmid using Phusion Polymerase(Thermo) following the manufacturer's instructions (two step protocol:98° C. for 10 sec, 72° C. for 45 sec) with the primers T1(/5Phos/GCAGCCAACTCAGCTTCTGCAGGGGCTTTGTTAGCAGCCGGATCCTC; SEQ ID NO:11)and T2 (AAACAAGCGCTCATGAGCCAGAAATCTGGAGCCCGATCTTCCCCATCGG; SEQ IDNO:12). The PCR product was digested with PstI to generate a 3′ overhangon one side, that was then tailed with ddTTP using TdT to preventelongation of the generated 3′ overhang. After tailing, the DNA wasdigested with BstXI to generate a 3′ overhang on the other end of theamplicon to enable extensions by polymerase-nucleotide conjugates. Thedigestion product was isolated by 2% agarose gel electrophoresis andpurified to yield the initial substrate for extensions bypolymerase-nucleotide conjugates.

The extension reactions were performed for 90 sec at 37° C. in 1×RBCwith 1 mg/mL of the respective polymerase-nucleotide conjugate and werequenched by the addition of an equal volume of quenching buffer (250 mMEDTA, 500 mM NaCl). Cleavage of the linker was performed by irradiationat 365 nm. The first extension reaction contained ˜40 nM of the initialsubstrate. After each cleavage step, the DNA products were purified(Zymo OCC), and the recovered DNA was subjected to the next extensionstep. The following conjugates were used in the extension steps: 1)TdT-dCTP, 2) TdT-dTTP, 3) TdT-dATP, 4) TdT-dCTP, 5) TdT-dTTP, 6)TdT-dGTP, 7) TdT-dATP, 8) TdT-dCTP, 9) TdT-dTTP, 10) TdT-dGTP. Theten-cycle product was T-tailed using TdT and free dTTP+ddTTP at a ratioof 100:1 and acetylated using 20 mM NHS-acetate in bicarbonate buffer.The tailed product was then PCR-amplified using HotStart Taq (NEB) withprimers C3 and C4 (GTGCCGTGAGACCTGGCTCCTGACGAGGAtaagcttCTATAGTGAGTCGTATTAATTTCG; SEQ ID NO:13) (PCR program: Initial cycle of 98° C. for 2min, 49° C. for 20 sec, 68° C. for 12 min, then 30 cycles of three stepprotocol: 98° C. for 30 sec, 49° C. for 20 sec, 68° C. for 30 sec). ThePCR product was inserted into pUC19 using EcoRI and HindIII sites thatwere introduced by the PCR primers. The plasmids were transformed intoDH10B cells and the plasmids of single colonies were extracted afterovernight growth in LB and sequenced.

As described in more detail above, a double stranded DNA template waselongated by 10 cycles using polymerase-nucleotide conjugates, thesynthesis product was amplified and cloned for sequencing (FIG. 14A). Of32 clones sequenced, 13 (41%) contained the complete 5′-CTACTGACTG-3′sequence (FIG. 14B), implying an average stepwise yield of 91%.

This result demonstrates that the cyclic process of DNA extension can berepeated many times to write nucleic acid molecules of the desiredsequence and length.

The invention claimed is:
 1. A conjugate comprising a nucleotide, alinker, and a polymerase capable of catalyzing the covalent addition ofthe nucleotide onto the 3′ end of a nucleic acid, wherein the linkertethers the nucleotide to the polymerase, and wherein the linker isbound to the nucleotide at the nucleobase, sugar, or the α-phosphate ofthe nucleotide.
 2. The conjugate of claim 1, wherein the linker isselectively cleavable.
 3. The conjugate of claim 2, wherein cleavage ofthe linker releases the polymerase from the nucleotide.
 4. The conjugateof claim 3, wherein cleavage of the linker leaves a scar on thenucleobase of the nucleotide, and wherein the scar comprises a part ofthe linker retained on the nucleobase after cleavage of the linker. 5.The conjugate of claim 4, wherein the scar on the nucleobase isselectively removable.
 6. The conjugate of claim 5, wherein removal ofthe scar leaves a naturally occurring nucleobase.
 7. The conjugate ofclaim 1, wherein the nucleotide is a nucleoside triphosphate.
 8. Theconjugate of claim 1, wherein the nucleotide comprises a nucleobaseselected from the group consisting of adenine, cytosine, guanine,thymine, and uracil.
 9. A method of nucleic acid synthesis, comprising:providing a conjugate comprising a nucleotide, a linker, and apolymerase capable of catalyzing the covalent addition of the nucleotideonto the 3′ end of a nucleic acid, wherein the linker tethers thenucleotide to the polymerase, and wherein the linker is bound to thenucleotide at the nucleobase, the sugar, or the α-phosphate of thenucleotide; and contacting a sample comprising the nucleic acid with theconjugate.
 10. The method of claim 9, wherein the linker is selectivelycleavable.
 11. The method of claim 10, further comprising exposing saidsample to conditions to cleave the linker to separate the polymerasefrom the nucleotide.
 12. The method of claim 9, wherein said polymerasecatalyzes the covalent addition of the nucleotide onto the 3′ end of thenucleic acid to form an extended polynucleotide.
 13. The method of claim12, further comprising cleaving said linker after said nucleotide ofsaid conjugate is covalently bound to the 3′ end of the nucleic acid.14. The method of claim 13, wherein the cleavage of the linker releasesthe polymerase from the extended polynucleotide.
 15. The method of claim13, wherein the extended polynucleotide comprises a scar followingcleavage of the linker, and wherein the scar comprises a part of thelinker retained on the nucleobase after cleavage of the linker.
 16. Themethod of claim 15, wherein the scar is selectively removable.
 17. Themethod of claim 15, further comprising removing said scar from saidextended polynucleotide.
 18. The method of claim 17, wherein saidextended polynucleotide comprises only naturally occurring nucleobasesafter removal of said scar.
 19. The method of claim 9, wherein saidnucleotide is a nucleoside triphosphate before covalent addition to the3′ end of the nucleic acid.
 20. A method of synthesizing apolynucleotide having a defined sequence, comprising steps: (a)contacting a substrate comprising a nucleic acid with a conjugatecomprising nucleotide, a linker, and a polymerase capable of catalyzingthe covalent addition of the nucleotide onto the 3′ end of a nucleicacid, wherein the linker tethers the nucleotide to the polymerase, andwherein the linker is bound to the nucleotide at the nucleobase, thesugar, or the α-phosphate of the nucleotide; (b) exposing said substrateto conditions sufficient to cleave the linker to separate the polymerasefrom the nucleotide; and (c) repeating steps (a) and (b) one or moretimes to synthesize the polynucleotide having a defined sequence. 21.The method of claim 20, wherein the linker is selectively cleavable. 22.The method of claim 20, wherein said polymerase catalyzes the covalentaddition of the nucleotide onto the 3′ end of the nucleic acid.
 23. Themethod of claim 22, wherein said linker is cleaved after the covalentaddition of the nucleotide onto the 3′ end of the nucleic acid.
 24. Themethod of claim 23, wherein the cleavage of the linker releases thepolymerase from covalently bound nucleotide.
 25. The method of claim 23,wherein the nucleic acid comprises a scar following cleavage of thelinker, and wherein the scar comprises a part of the linker retained onthe nucleobase after cleavage of the linker.
 26. The method of claim 25,wherein the scar is selectively removable.
 27. The method of claim 25,further comprising removing said scar from said nucleic acid.
 28. Themethod of claim 27, wherein nucleic acid comprises only naturallyoccurring nucleobases after removal of said scar.
 29. The method ofclaim 20, wherein said nucleotide is a nucleoside triphosphate beforecovalent addition to the 3′ end of the nucleic acid.
 30. A method ofnucleic acid synthesis, comprising: providing a conjugate comprising anucleotide, a linker, and a polymerase capable of catalyzing thecovalent addition of the nucleotide onto the 3′ end of a nucleic acid,wherein the linker tethers the nucleotide to the polymerase, and whereinthe linker is bound to the nucleotide at the nucleobase, the sugar, orthe α-phosphate of the nucleotide; contacting a sample comprising thenucleic acid with the conjugate, wherein said polymerase catalyzes thecovalent addition of the nucleotide onto the 3′ end of the nucleic acidto form an extended polynucleotide; cleaving said linker after saidnucleotide of said conjugate is covalently bound to the 3′ end of thenucleic acid, wherein said cleavage leaves a scar on the nucleobase ofsaid covalently bound nucleotide; and removing said scar from saidnucleobase.